s41586-024-08276-1

Article
The oestrous cycle stage affects mammary

tumour sensitivity to chemotherapy
https://doi.org/10.1038/s41586-024-08276-1 Laura Bornes1, Lennart J. van Winden2, Veerle C. M. Geurts3, Beaunelle de Bruijn4,
Leyla Azarang5, Mirthe Lanfermeijer2, Marika Caruso4, Natalie Proost6, Manon Boeije6,
Received: 24 August 2021
Jeroen O. Lohuis1, Guillaume Belthier1, Eulàlia Noguera Delgado1, Nadia de Gruil7,
Accepted: 23 October 2024 Judith R. Kroep7, Marieke van de Ven6, Renee Menezes5, Jelle Wesseling1,8,9, Marleen Kok3,10,
Sabine Linn1,10,11, Annegien Broeks12, Huub H. van Rossum2, Colinda L. G. J. Scheele4,13 ✉ &
Published online: xx xx xxxx
Jacco van Rheenen1,13 ✉
Open access
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The response of breast cancer to neoadjuvant chemotherapy (NAC) varies
substantially, even when tumours belong to the same molecular or histological
subtype1. Here we identify the oestrous cycle as an important contributor to this
heterogeneity. In three mouse models of breast cancer, we show reduced responses
to NAC when treatment is initiated during the dioestrus stage, when compared with
initiation during the oestrus stage. Similar findings were observed in retrospective
premenopausal cohorts of human patients. Mechanistically, the dioestrus stage
exhibits systemic and localized changes, including (1) an increased number of cells
undergoing epithelial-to-mesenchymal transition linked to chemoresistance2–4 and
(2) decreased tumour vessel diameter, suggesting potential constraints to drug
sensitivity and delivery. In addition, an elevated presence of macrophages, previously
associated with chemoresistance induction5, characterizes the dioestrus phase.
Whereas NAC disrupts the oestrous cycle, this elevated macrophage prevalence
persists and depletion of macrophages mitigates the reduced therapy response
observed when initiating treatment during dioestrus. Our data collectively
demonstrate the oestrous cycle as a crucial infradian rhythm determining
chemosensitivity, warranting future clinical studies to exploit optimal treatment
initiation timing for enhanced chemotherapy outcomes.
The body contains several internal timekeeping clocks to deal with oscil- Neoadjuvant chemotherapy (NAC) is currently offered to patients
lating environmental changes, such as variations in light and tempera- with locally advanced breast cancer (BC) to eliminate potential micro-
ture. Over a 24 h period, physiological adaptations are orchestrated by metastases, and to those who would benefit from size reduction of
the well-documented circadian rhythm6. For cycles exceeding 24 h, the the primary tumour before undergoing breast-conserving surgery1,15.
body contains distinct infradian rhythms7. The menstrual cycle—and However, the effectiveness of NAC is highly heterogeneous, even for
its murine equivalent, the oestrous cycle—are a typical example of tumours with the same molecular or pathological subtype. This vari-
such an infradian rhythm that regulates alternating periods of fer- ation led us to explore whether the oestrous/menstrual cycle might
tile and non-fertile phases in females. This oestrous/menstrual cycle partly explain differences in the effectiveness of NAC. During different
is regulated by fluctuating hormone levels that are produced by the phases of the menstrual cycle, the epithelium in breast lobules under-
hypothalamic–pituitary–gonadal axis. These hormonal fluctuations goes major histological changes16,17. Analogous morphological changes
are accompanied by local alterations, such as remodelling of the uterus have been observed in the murine mammary epithelium, including
and mammary tissue, and systemic alterations, such as changes in the formation and regression of alveolar buds during the oestrous
body temperature, dilation and constriction of blood vessels, and by cycle18,19. Nevertheless, it remains to be determined whether these
differences in the immune landscape8–10. Internal rhythms that regu- distinct phases of cellular turnover observed in healthy ducts20–22 are
late large-scale cyclic alterations enable the body to adapt to internal maintained in tumours. Moreover, it is unknown whether these local or
and external changes, potentially including response to therapies11–14. systemic changes during the oestrous/menstrual cycle can be a source
1
Division of Molecular Pathology, Oncode Institute, The Netherlands Cancer Institute, Amsterdam, the Netherlands. 2Laboratory of Clinical Chemistry and Hematology, The Netherlands Cancer
Institute, Amsterdam, the Netherlands. 3Division of Tumor Biology & Immunology, The Netherlands Cancer Institute, Amsterdam, the Netherlands. 4VIB Center for Cancer Biology, KU Leuven
Department of Oncology, Leuven, Belgium. 5Biostatistics Centre & Department of Psychosocial Research and Epidemiology, The Netherlands Cancer Institute, Amsterdam, the Netherlands.
6
Mouse Clinic for Cancer and Aging (MCCA) Preclinical Intervention Unit, The Netherlands Cancer Institute, Amsterdam, the Netherlands. 7Department of Medical Oncology, Leiden University
Medical Center, Leiden, the Netherlands. 8Department of Pathology, The Netherlands Cancer Institute – Antoni van Leeuwenhoek, Amsterdam, the Netherlands. 9Department of Pathology,
Leiden University Medical Center, Leiden, the Netherlands. 10Department of Medical Oncology, The Netherlands Cancer Institute, Amsterdam, the Netherlands. 11Department of Pathology,
Utrecht University Medical Center, Utrecht, the Netherlands. 12Core Facility Molecular Pathology & Biobanking, The Netherlands Cancer Institute, Amsterdam, the Netherlands. 13These authors
contributed equally: Colinda L.G.J. Scheele, Jacco van Rheenen. ✉e-mail: colinda.scheele@kuleuven.be; j.v.rheenen@nki.nl
Nature | www.nature.com | 1
Article
for the observed heterogeneity in response to NAC, and whether they tumours resembling invasive BC30. In this model, we observed similar
can be leveraged to improve NAC treatment outcomes23. clonal growth and regression patterns by multiday IVM (Extended
Data Fig. 2a,b). Furthermore, we found that proliferation levels in
WNT1-driven mammary tumours were higher during the oestrus stage
Oestrous cycle-driven tumour cell turnover compared with dioestrus (Extended Data Fig. 2c).
To prepare the healthy murine mammary epithelium for a possible
pregnancy, proliferation drives the formation of alveolar buds dur-
ing the oestrus stage. In the absence of pregnancy, however, these NAC responses vary during the oestrous cycle
alveolar buds regress following coordinated cell death during the Neoadjuvant chemotherapy is the preferred treatment of choice for
dioestrus stage18,22 (Extended Data Fig. 1a). Previous research showed locally advanced BC, aimed at elimination of micrometastases and
that this cycle takes 4–6 days and that it can be blocked following ova- tumour debulking, thereby increasing the likelihood of breast- and
riectomy24,25 (Extended Data Fig. 1a). axilla-conserving surgery. The established chemotherapeutics, such as
To explore whether mammary tumours exhibit similar cyclic pat- anthracyclines (for instance, doxorubicin), cyclophosphamide and car-
terns, we studied the behaviour of individual tumour cell clones in the boplatin, are aimed at targeting rapidly proliferating cells31. Therefore,
MMTV-PyMT mouse model of invasive BC26 by intravital microscopy (IVM) we hypothesized that chemotherapy might be more effective in treat-
(Fig. 1a). For visualization of individual clones, we crossed MMTV-PyMT ing BC during the more proliferative oestrus stage than in dioestrus.
mice with R26-CreERT2het;R26R-Confettihet mice. Following mammary To test this hypothesis, we administered doxorubicin or cyclophos-
tumour development, individual tumour cells were fluorescently labelled phamide to MMTV-PyMT;MMTV-Cre;R26-LSL-YFP;E-cad-CFP female mice
by administration of a low dose of tamoxifen (1.5 mg 25 g−1 body weight), bearing tumours with a cumulative volume of 500–750 mm3. We initi-
resulting in stochastic Cre-mediated recombination of the confetti cas- ated treatment at either the oestrus or dioestrus stage. Tumours were
sette in a minority of tumour cells throughout the tumour mass, and dissected 2–4 days later, and the level of cell death in these tumours was
subsequent expression of one of the four confetti colours (yellow fluo- determined using flow-cytometric analysis (Fig. 2a and Extended Data
rescent protein, green fluorescent protein, red fluorescent protein and Fig. 3a). Notably, this analysis showed that treatment with doxorubicin
cyan fluorescent protein (CFP)) as an inheritable mark (Extended Data or cyclophosphamide during oestrus resulted in a significantly higher
Fig. 1b). Next, a mammary imaging window was implanted over the mam- level of cell death when compared with treatment during dioestrus
mary tumour between 7 and 14 days after confetti colour induction, as (Fig. 2b), which was corroborated by a marginally significant reduction
described in ref. 27. Using multiday IVM of the same tumour area through in tumour burden (Fig. 2c).
the mammary imaging window, we followed individual tumour cell We next questioned whether the increased NAC sensitivity observed
clones over time (Fig. 1a,b). Interestingly, a subset of clones showed syn- during the oestrus stage would persist across successive treatments. To
chronized expansion and regression (Fig. 1c and Extended Data Fig. 1c). this end, we initiated NAC when mice had a cumulative tumour volume
To test whether this synchronous expansion and regression mirrored the of 250–450 mm3 at either the oestrus or dioestrus stage. Mice underwent
oestrous cycle-dependent growth and regression patterns occurring five consecutive chemotherapy treatments, until either their cumula-
in the disease-free mammary gland, we impeded the oestrous cycle by tive tumour volume exceeded 750 mm3 or a single tumour exceeded
ovariectomy. As anticipated, this procedure decreased the coordinated 350 mm3 (Fig. 2d). Given that chemotherapy often disrupts the oestrous/
phases of expansion and regression of tumour cell clones (Fig. 1c,d and menstrual cycle32–38, we first questioned whether this holds true in our
Extended Data Fig. 1c). These findings suggest that the specific oestrous experiments. Postchemotherapy vaginal smears showed a disrupted
cycle-dependent cellular turnover identified in disease-free ducts is, to oestrous cycle (presence of abnormal cells, absence of cells, stalling
a certain extent, conserved within mammary tumours. of changes in cycle-specific cells) in the majority of mice (Fig. 2e and
To validate these observations using an independent method, Extended Data Fig. 3b). These findings were corroborated by a reduc-
we labelled proliferative tumour cells using 4 h pulses of 5-ethynyl- tion in preantral follicle count in the ovaries (Fig. 2f and Extended Data
2′-deoxyuridine (EdU), a marker for DNA replication, and phospho- Fig. 3c–e), in accordance with previous reports37,38. In addition, we
histone H3 (PHH3), a marker for mitosis. We determined oestrus stage observed a significant reduction in serum progesterone levels, affirm-
by vaginal cytology, as previously established28 (Extended Data Fig. 1d). ing the profound impact of chemotherapy on the mouse reproductive
The accuracy of our staging approach was confirmed by determination cycle (Fig. 2g). Notably, regardless of the disrupted oestrous cycle, the
of serum progesterone levels in mice at different stages (Extended difference in treatment response was maintained over the long term
Data Fig. 1e). On the basis of staging results, we harvested tumours of (Fig. 2h, left). To test for significance, we used a generalized-nested,
comparable tumour size at either the oestrus or dioestrus stage. Next, mixed-effects model on fitted tumour volumes for both oestrus and
we determined the location of proliferative cells within the tumour. In dioestrus stages, which showed significant separation between phases
accordance with previous findings in intestinal tumours29, we found (Fig. 2h, middle). Moreover, the superior response in mice that instigated
that the majority of proliferation occurred at the edges of mammary NAC treatment during the oestrus phase was also reflected in extended
tumour lobes, and we did not observe spatial differences between overall survival (Fig. 2h, right). Indeed, in line with a higher response
different stages of the oestrous cycle (Extended Data Fig. 1f). On the in the primary tumour, metastatic load in the lungs was significantly
basis of previous IVM experiments, we expect that proliferation in reduced in mice that began NAC during the oestrus stage compared
tumour tissues should be higher in the oestrus than in the dioestrus with those that started treatment during the dioestrus stage (Fig. 2i,j).
stage, and indeed we observed a significantly increased number of cells Important to note here is that primary tumour sizes at endpoint did not
that went through S-phase during the oestrus stage when compared differ significantly between oestrous and dioestrous mice, and lungs
with dioestrus (Fig. 1e). We confirmed this result using an independent from the oestrus-initiated treatment group were even analysed at a
approach, scoring the mitotic index on the basis of PHH3 staining dur- later time point owing to the extended survival of these mice (Fig. 2k–l).
ing the oestrus and dioestrus stages (Fig. 1f). Increased proliferation Collectively, these data demonstrate the impact of the oestrous cycle
levels did not correlate with the expression level of the PyMT oncogene on both the immediate and prolonged NAC response in PyMT tumours.
(Extended Data Fig. 1g), excluding the possibility that differences in
proliferation are caused by heterogeneity in PyMT expression. To fur-
ther validate these observations in a separate mouse model of BC, we Reduced NAC response in the dioestrus stage
performed similar experiments using the MMTV-Wnt1 mouse model, To explore whether the loss of expression of hormone receptors (oestro-
in which constitutive WNT1 overexpression leads to the formation of gen receptor (ER) and progesterone receptor (PR)) negates the impact
2 | Nature | www.nature.com
a b
Imaging session 1 Clone 1 Clone 2
Overview image
Clone size (number of cells)

2 400
Day 0
1 300
200
100
Day 3
0
0 3 6 9 12
Confetti
Second harmonic generation Days of lineage tracing
Imaging session 2
101
Overview image Growth
Day 6
Clone growth rate

2
1
100
Regression
10–1
Day 9
0 3 6 9 12
Days of lineage tracing
Confetti
Second harmonic generation
c Non-ovariectomized Ovariectomized
102 Growth 102 Growth 102 Growth 102 Growth
I II I II
Clone growth rate
Clone growth rate
Clone growth rate
Clone growth rate

101 101 101 101
100 100 100 100
10–1 10–1 10–1 10–1

Regression Regression Regression Regression
10–2 10–2 10–2 10–2
0 5 10 0 5 10 0 5 10 0 5 10
Days of lineage tracing Days of lineage tracing Days of lineage tracing Days of lineage tracing
d e f DNA Tumour cells

100 E-cadCFP PHH3
* DNA EdU 15 * **
3
PHH3+ tumour cells per mm2

Clones with synchronized
proliferation/cell death
80
Oestrus
Oestrus
EdU+ cells (%)
10
phases (%)
60 2
40
5 1
20
Dioestrus
Dioestrus
0 0 0
s
s
s
s
trl
VX
tru
ru
tru
ru
C
st
st
O
es
es
oe
oe
O
O
Di
Di
Fig. 1 | Oestrous cycle-dependent proliferation of tumour cells during alternating growth rates, as determined by multiday IVM for physiological
oestrus stage in MMTV-PyMT tumours. a, Schematic representation of cycling mice (Ctrl; n = 5 mice) and ovariectomized mice (OVX; n = 4 mice).
multiday IVM set-up (left), with representative overview images (single z-plane) e,f, Representative immune fluorescent images and quantification of EdU
(right) of five mice in the first imaging session (top) and the following imaging incorporation (e) and expression of PHH3 (f) in genetic MMTV-PyMT mammary
session (bottom). Rectangles indicate example clones represented over tumours during different stages of the oestrous cycle (n = 3 mice per oestrous
multiple imaging days shown in b (left). b, Total clone size (number of cells in stage, 14 and 11 individual tumours for EdU and PHH3 staining, respectively).
a clone) (right, top) and clone growth rate (right, bottom) of example clones d–f, Thicker lines represent median, thinner lines indicate the 25th and 75th
quantified over time. c, Representative examples of clone growth rate in percentiles and dots represent individual tumours. Statistical analysis by
genetic MMTV-PyMT tumours imaged over multiple days with IVM. Graphs two-sided Wilcoxon–Mann–Whitney test (d) or one-sided hypothesis testing
depict all growth rates (graph I) or a selection of clones (graph II) measured in using linear mixed-effects model (e,f). *P < 0.05, **P < 0.01. Further details on
physiologically cycling mice (left) and ovariectomized mice (right). Each line statistical analysis are provided in Supplementary File 1. Scale bars, 500 µm (a),
represents a clone, and coloured lines highlight the behaviour of individual 100 µm (b,e,f).
clones. d, Violin plots depicting percentage of clones showing synchronized
of oestrous cycle stage on NAC responses, we conducted parallel experi- our findings in the PyMT tumour model, the hormone receptor-positive
ments in both hormone receptor-positive (MMTV-Wnt1) and hormone MMTV-Wnt1 model showed that mice receiving an initial NAC dose
receptor-negative (Brca1−/−;Trp53−/−) BC models (Fig. 3a–d). Mirroring during the oestrus stage demonstrated a sustained enhanced response
Article
a b Doxorubicin Cyclophosphamide
c Doxorubicin Cyclophosphamide
* * # #
50 50 100 100
Time of
treatment
change from baseline (%)

Apoptotic cells (PI+) 40 40
PI+ tumour cells (%)

(flow cytometry) 50 50
Tumour burden
Tumour burden
Stage:
O or D D0 30 30
0 0
20 20
Measurement of tumour volume –50 –50

10 10
(change from baseline)
0 0 –100 –100
s
s
s
s
s
s
ru
tru
tru
ru
ru
tru
ru
ru
st
t
st
st
st
es
es
es
es
oe
oe
oe
oe
O
O
O
Di
Di
Di
Di
Stage Stage Stage Stage
at treatment at treatment at treatment at treatment
d Time of treatment f g
Follicle count Hormone measurement
Stage:
O or D D0 D7 D14 D21 D28 Oestrus Dioestrus Oestrus Dioestrus
20 20 100 100
* *** * **
Progesterone (nmol l–1)
Progesterone (nmol l–1)

80 80
Measurement of tumour volume 15 15
Follicles per mm2

Follicles per mm2
Staging (vaginal smear)

Follicle count 60 60
Hormone measurement 10 10
40 40
e Staging
5 5
Oestrus Dioestrus 20 20
0 0 0 0
Disrupted Doxo – + + Doxo – + + Doxo – + + Doxo – + +
Cycling Time 0 7 LT Time 0 7 LT Time 0 7 LT Time 0 7 LT
h
Stage at first treatment: Oestrus Dioestrus
Time of treatment 200 Oestrus Time of treatment

Dioestrus **
100
Tumour volume (mm3)
(normalized to baseline)
Overall survival (%)
2 150
Tumour volume
50
1 100
Oestrus
Dioestrus **
0 50 0
0 5 10 15 20 25 0 10 20 30 0 10 20 30
Time after first treatment (days) Time after first treatment (days) Time after first treatment (days)
i j k NS l #
60 * 800 40
Oestrus Dioestrus
Number of metastatic foci
first treatment (days)
600 30
Time elapsed after
at endpoint (mm3)
Tumour volume
40
400 20
Lung metastases
20
200 10
0 0 0
s
s
Di rus
s
s
s
tru
tru
ru
tru
ru
t
st
st
es
es
es
s
oe
oe
oe
O
O
O
Di
Di
Stage Stage Stage

at treatment at treatment at treatment
Fig. 2 | Decreased sensitivity to NAC in MMTV-PyMT tumours during the point at which over 40% of mice reached humane endpoint. Middle,
dioestrus. a, Experimental set-up; stages: oestrus (O) and dioestrus (D). fitted growth curves of mixed-effects model representing tumour volume.
b,c, Percentage of PI+ tumour cells (b) and change in tumour burden (c). Right, Kaplan–Meier survival analysis; n: O = 8, D = 9 mice. i. Representative
n: ODoxo = 5 mice/30 tumours; DDoxo = 3 mice/22 tumours; OCyclo = 6 mice/47 immunofluorescent lung section images, arrows indicate metastatic nodules.
tumours; DCyclo = 5 mice/40 tumours. d, Experimental set-up. e, Proportion j–l, Quantification of macrometastatic nodules ( j), tumour volume (k) and
of mice exhibiting physiological or disrupted oestrous cycle following time elapsed between first treatment and analysis (l); n = 5 mice. b,c,f,g,j,k,
doxorubicin treatment initiated at the indicated stage; n: O = 13, D = 10 mice. Thicker solid lines represent median, and thinner solid lines the 25th and 75th
f,g, Number of preantral follicles per square millimetre in ovaries (f) and percentiles. Individual dots represent either a tumour (b) or a mouse (c,f,g,h,
progesterone levels in serum (g) at pretreatment, 7 days following initial j–l). Statistical analysis by either one-sided hypothesis testing using linear
treatment and long term (LT) at the endpoint of experiment. Follicle count, mixed-effects model (b), one-sided Wilcoxon–Mann–Whitney (c,j), Kruskal–
n: O0 = 10; O7 = 7; OLT = 4; D0 = 10; D7 = 7; DLT = 8 mice. Hormone measurements, Wallis (f,g), nonlinear modelling of tumour growth (h, left), a joint model
n: O0 = 9; O7 = 10; OLT = 8; D0 = 10; D7 = 7; DLT = 9 mice. h, Left, effect of oxorubicin (h, middle), a regression model (h, right) or two-sided Wilcoxon–Mann–Whitney
treatment. Symbols represent mean relative tumour size per mouse; n = 8–9 test (k,l). #P < 0.1, *P < 0.05, **P < 0.01, ***P < 0.001. Further details on statistical
mice per group, O = 69 and D = 84 tumours at first treatment. Lines represent analysis are provided in Supplementary Files 1 and 2. D0, day 0; NS, not
mean of all tumours, shading represents s.e.m. Treatment effect is shown until significant; PI, propidium iodide. Scale bars, 2 mm.
WNT1
a b
Stage at first treatment: Oestrus Dioestrus
WNT1
ER PR
Time of treatment Time of treatment
Normalized treatment effect

800 Oestrus
*
Oestrus
Tumour volume (mm3)

Dioestrus 100
2 Oestrus
600 Dioestrus
#

400
1 50
Dioestrus
200
0 0 0
0 5 10 0 10 20 30 0 10 20 30
c d Brca1–/–Trp53–/–
Brca1–/–Trp53–/– Stage at first treatment: Oestrus Dioestrus

ER PR
Time of treatment Time of treatment
400 Oestrus NS
Dioestrus
100
Oestrus
Tumour volume (mm3)

2 300

200
50
1
Dioestrus
100
Oestrus
Dioestrus *
0 0 0
0 10 20 30 40 0 20 40 60 0 20 40 60
Transplanted PyMT
e f g
Stage at first treatment: Oestrus Dioestrus OVX
Genetic PyMT Transplanted PyMT

Time of treatment Sham oestrus
ER PR ER PR Sham dioestrus ** **
4 2 OVX **
Oestrus
Tumour volume (cm3)

Oestrus
2 1
Dioestrus
1 Sham oestrus
Dioestrus
Sham dioestrus
OVX
0 0
0 10 20 30 0 20 40 60 80
Time after first treatment (days) Time after first treatment (days)
Fig. 3 | Reduced chemosensitivity during dioestrus in hormone MMTV-Wnt1 (b) and Brca1−/−;Trp53 −/− (d) mice treated with doxorubicin initiated
receptor-positive and hormone receptor-negative tumour models. during oestrus or dioestrus. MMTV-Wnt1: oestrus, n = 6 mice; dioestrus,
a,c,e,f, Immunohistochemical staining for ER (left) and PR (right), n = 5 mice; Brca1−/−;Trp53 −/−: oestrus, n = 6 mice; dioestrus n = 8 mice. g, Left,
representative of more than five treatment-naive tumours in more than five treatment effect in transplanted MMTV-PyMT model, represented as tumour
mice of the genetic MMTV-Wnt1 (a), transplanted Brca1−/−;Trp53 −/− (c) and size normalized to initial treatment size. Arrows indicate rounds of treatment,
genetic (e) and transplanted MMTV-PyMT (f) models at oestrus (top) and lines the tumour means and shading s.e.m.; n = 8–13 mice per group, with
dioestrus (bottom). b,d, Left, treatment effect in transplanted MMTV-Wnt1 O = 25, D = 15 and OVX = 20 tumours at first treatment. Right, fitted growth
(b) and Brca1−/−;Trp53 −/− (d) models, represented as tumour size normalized to curves of mixed-effects model representing tumour volume. b,d,g, Left,
initial treatment size. Arrows indicate rounds of treatment, lines the tumour significance was determined using nonlinear modelling of tumour growth.
means and shading s.e.m. MMTV-Wnt1 model: n = 5–6 mice per group with O = 6 Treatment effect is shown until over 40% (b,g) or over 20% (d) of mice reached
and D = 6 tumours at first treatment. Brca1−/−;Trp53 −/− model: n = 6–8 mice per humane endpoint. Response between groups was statistically evaluated
group, with O = 12 and D = 14 tumours at first treatment. Middle, fitted growth using a joint model (b,d,g, middle); survival analysis was performed using a
curves of mixed-effects model representing tumour volume for transplanted regression model (b,d,g, right). #P < 0.1, *P < 0.05; **P < 0.01. Scale bars, 150 µm.
MMTV-Wnt1 (b) and Brca1−/−;Trp53 −/− (d) models. Right, Kaplan–Meier curves for
during successive treatments, when compared with those that began a significant difference in survival was observed (Fig. 3d, right). This
NAC treatment during the dioestrus stage (Fig. 3b). Unexpectedly, implies that NAC sensitivity tied to the oestrous stage might be inde-
we also obtained similar data in the hormone receptor-negative pendent of hormone receptor expression by tumour cells.
Brca1−/−;Trp53−/− model. Despite minor differences in tumour reduc- To test this idea, we performed experiments in a hormone receptor-
tion (Fig. 3d, left and middle), indicative of a good overall response, negative PyMT tumour model by transplanting late-stage PyMT tumour
Article
cells into the mammary fat pad of FVB mice. Although PyMT tumours, a lower number of proliferative cells and reduced vasoconstriction
at their early stages of development, express hormone receptors39, (potentially leading to reduced drug delivery), an increased number
these tumours progressively lose hormone receptor expression dur- of more chemoresistant mesenchymal tumour cells and an increase
ing tumour progression (Fig. 3e,f). In line with data from the hor- in macrophage abundance during the dioestrus phase. Importantly,
mone receptor-negative Brca1−/−;Trp53−/− model, a marked difference depletion of macrophages led to similar treatment effects in mice
in tumour response was observed in transplanted PyMT tumours, treated in both oestrus and dioestrus, indicating that the increased
depending on whether NAC treatment was started in the oestrus or chemoresistance is, at least in part, mediated by their presence. The
dioestrus phase (Fig. 3g). This confirms that the influence of the oes- sustained increase in macrophages following the first treatment in
trous cycle on NAC responses may be independent of hormone recep- dioestrus explains why the therapy-suppressive phenotype persists
tor expression. To further challenge this idea, we performed the same even when the physiological oestrous cycle is lost.
experiment in PyMT tumour-bearing ovariectomized mice. Interest-
ingly, NAC outcomes in these ovariectomized mice closely mirrored
the effect observed when NAC was initiated during the oestrus stage, Menstrual cycle impacts NAC response in BC
and markedly surpassed the treatment response with NAC initiated at To investigate whether our findings in mouse models of BC translate to
the dioestrus stage (Fig. 3g). This observation further reinforces the the human setting, we performed a retrospective study using patient
idea that hormone receptor-mediated proliferation status may not data from the Antoni van Leeuwenhoek Hospital (Amsterdam) and
be the main determinant of chemosensitivity. Moreover, in contrast the multicentre Dutch Breast Cancer Research Group DIRECT trial45.
to the notion of increased sensitivity during the oestrus stage, these Whereas the oestrous cycle in rodents spans only 4–6 days46, in women
data suggest that mice in the dioestrus stage may experience reduced it spans approximately 1 month and involves menstruation (that is,
chemosensitivity. a menstrual cycle). During the menstrual cycle, serum levels of the
steroid hormone progesterone mark distinct phases: progesterone
levels are low during the follicular phase (below 2.2 nmol l−1), rise during
Mechanisms of reduced NAC response the mid-cycle phase (2.2–6.4 nmol l−1) and peak during the luteal phase
We next explored potential mechanisms that can contribute to the (above 6.4 nmol l−1)47. Therefore, to investigate potential differential
reduced sensitivity of NAC when initiated in the dioestrus stage. responses at distinct phases of the menstrual cycle, we used proges-
A rich body of literature describes many factors that can contribute terone concentrations in biobanked serum as a proxy for different
to reduced chemosensitivity, including epithelial-to-mesenchymal phases of the cycle.
transition (EMT)2–4 and changes in the tumour microenvironment, For the first cohort (cohort 1), we selected premenopausal patients
specifically the immune landscape5,40–42. (under 50 years of age) with hormone receptor (HR)+ and human
To explore whether the EMT state of tumour cells could explain the epidermal growth factor receptor 2 (HER2)− (HR+HER2−) BC (Fig. 5a).
variation in sensitivity to NAC observed during oestrous cycle stages, To focus solely on patients with a physiological menstrual cycle, we
we used our previously established EMT fluorescent reporter PyMT excluded those using hormonal contraception, pregnant patients and
mouse model (MMTV-PyMT;MMTV-Cre;R26-LSL-YFP;E-cad-mCFP)43,44. In those who had undergone oophorectomy (Fig. 5a). Finally, we selected
this model, all cancer cells are fluorescently labelled with yellow fluores- from these patients only those who had a sufficient amount of serum
cent protein, whereas endogenous E-cadherin (E-cad) is conjugated to in the biobank (Fig. 5a). To obtain a reliable estimation of the status
monomeric CFP, facilitating the distinction between epithelial (E-cadHI) of the menstrual cycle at the time of therapy initiation, we included
and mesenchymal (E-cadLO) cancer cell states43. As we have previously only patients whose serum was biobanked within 5 days preceding
shown, E-cadLO cells have a mesenchymal phenotype and expression NAC. Because individuals in the mid-cycle progress within 1–3 days
profile, including increased migratory behaviour and upregulation of to the luteal phase, patients in mid-cycle were categorized under the
mesenchymal markers such as vimentin43,44. Flow-cytometry analyses progesterone-high phase. Following these criteria, 17 and 13 patients
of treatment-naive tumours derived from this model showed a marked were determined as having started NAC treatment when progesterone
increase in mesenchymal cells (E-cadLO) during the dioestrus stage levels were low and high, respectively (Extended Data Fig. 6a). Crucially,
(Fig. 4a and Extended Data Fig. 3a). Because EMT is well known to lead known prognostic or predictive factors for NAC response1,15, including
to chemoresistance2–4, this increase might be a pivotal element behind age, receptor status, tumour size and tumour grade, showed balanced
the observed reduced NAC efficacy during the dioestrus stage. distribution across both groups (Extended Data Fig. 6b–f). Moreover,
Next, we examined the tumour microenvironment. First, we observed lymph node status and type of chemotherapy were similar between
a reduction in blood vessel diameter during the dioestrus stage groups (Extended Data Table 1).
(Fig. 4b), suggesting potential constraints in drug delivery. Second, Notably, similar to our findings of reduced sensitivity of NAC in the
during the dioestrus stage, we noted an increased presence of mac- dioestrus stage in mouse models, our retrospective analysis showed
rophages, but not of T cells, within treatment-naive tumours (Fig. 4c that progressive and steady disease was more frequently observed
and Extended Data Fig. 4a,b). This relative increase in macrophage among patients starting NAC in the progesterone-high phase com-
presence persisted, even 7 days post treatment when the oestrous cycle pared with those in the progesterone-low phase (Fig. 5b). In addition,
was disrupted (Fig. 4c). Given that macrophages can promote tumour partial and complete pathological responses were more prevalent
drug resistance (reviewed in ref. 5, for example), we explored their in the progesterone-low group (Fig. 5b). Moreover, we evaluated the
potential role in reduced chemotherapy sensitivity during the dioes- reduction of tumour burden by comparing tumour size in pretreatment
trus stage by antibody-mediated depletion (Fig. 4d–g and Extended clinical images with that of residual disease as assessed by pathological
Data Fig. 5). Indeed, macrophage depletion, but not T cell depletion, evaluation. Similar to our findings in mice, patients in which NAC was
enhanced therapy responses (that is, chemotherapy-induced cell death initiated during the progesterone-high phase had significantly lower
and tumour shrinkage) during the dioestrus stage to a level compa- tumour reduction compared with those in which NAC was initiated
rable to that during the oestrus stage (Fig. 4f,g and Extended Data during the progesterone-low phase (Fig. 5c,d).
Figs. 4c–f and 5). Because these data are based on a relatively small patient cohort,
Collectively, our mouse experiments show that the observed reduced we performed a second retrospective study in premenopausal
chemosensitivity in the dioestrus stage may be caused by a multitude of women with a physiological menstrual cycle who were diagnosed with
systemic and localized oestrous cycle-induced changes. As contribut- triple-negative breast cancer (TNBC). For this TNBC cohort, we main-
ing factors to the observed change in chemosensitivity, we identified tained the inclusion and exclusion criteria from the HR+HER2− cohort
a b CD31 Zoom
30 * 25 ***
intratumour vessels (Pm)

Mesenchymal E-cadLO
Oestrus
20
Average diameter of
tumour cells (%)
20
15
10
10
5
Dioestrus
0 0
Di rus
s
s
Di rus
ru
ru
t
st
st
t
es
es
oe
oe
O
O
c Pretreatment Posttreatment
F4/80 Zoom (7 days)
30 * 30 *
Oestrus
F4/80+ cells (%)
F4/80+ cells (%)

20 20
10 10
Dioestrus
0 0
s
tru
ru
tru
ru
st
st
es
es
oe
oe
O
O
Di
Di
Stage
at treatment
d e F4/80
Stage O or D (anti-IgG2a)
Apoptotic cells (PI+)
Control
(flow cytometry)
D7 D0
Measurement of tumour volume

(change from baseline)
(anti-CSF1R)
Macrophage
depletion
Treatment depletion Ab
Treatment doxorubicin
f Macrophage
g Macrophage
Control depletion Control depletion
(anti-IgG2a) (anti-CSF1R) (anti-IgG2a) (anti-CSF1R)
NS NS
50 * 50 100 * 100
40 40
50 50
Tumour burden
Tumour burden
30 30
0 0
20 20
–50 –50
10 10
0 0 –100 –100
s
Di rus
s
s
s
tru
tru
ru
tru
ru
ru
ru
st
st
st
st
es
es
es
es
oe
oe
oe
oe
O
O
Di
Di
Di
Stage Stage Stage Stage

at treatment at treatment at treatment at treatment
Fig. 4 | Mechanisms contributing to reduced chemosensitivity in dioestrus. (top, representative of 15 tumours/3 mice) or macrophage depletion antibody
a, Percentage of mesenchymal tumour cells determined in treatment-naive (Ab) (anti-CSF1R) (bottom, representative of 21 tumours/5 mice). f,g, Percentage
tumours of genetic MMTV-PyMT model at different oestrous cycle stages; n = 3 of PI+ tumour cells (f) and tumour volume change compared with baseline
mice/24 tumours per group. b, Left, imunohistochemical staining for CD31; (g) for mice receiving control antibody (anti-IgG2a) or macrophage depletion
right, quantified diameter of intratumoural vasculature in treatment-naive antibody (anti-CSF1R) and treatment with doxorubicin. n: Oa-IgG2a = 6 mice/24
genetic MMTV-PyMT tumours during stages of oestrous cycle. n = 3 mice/12 tumours; Da-IgG2a = 6 mice/28 tumours; Oa-CSF1R = 4 mice/17 tumours; Da-CSF1R = 5
tumours per oestrous stage. c, Left, immunohistochemical staining for F4/80 mice/21 tumours. a,b,c,f,g, Thicker solid lines represent median, and thinner
(left). Quantification of percentage of F4/80+ cells in treatment-naive tumours solid lines the 25th and 75th percentiles. Dots represent either individual
(middle) or 7 days post treatment (right) in the genetic MMTV-PyMT model at tumours (a–c,f) or mice (g). Statistical analysis was performed using a linear
oestrus and dioestrus. n: Opre = 5 mice/21 tumours; Dpre = 7 mice/27 tumours; mixed-effects model (a,c) or a two-sided (b) or one-sided (f,g) Wilcoxon–
Opost = 3 mice/12 tumours; Dpost = 3 mice/11 tumours. d, Schematic representation Mann–Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001. Further details on
of experimental set-up. e, Immunohistochemical staining of chemotherapy statistical analysis are provided in Supplementary File 1. Scale bars, 500 µm
treatment-naive tumours treated with either control antibody (anti-IgG2a) (b (left)), 50 µm (b (right), c (right)), 200 µm (c (left), e).
Article
a Cohort 1 Cohort 2
Patients with HR+HER2– BC in the Patients with TNBC in AvL-NKI-DB
AvL-NKI-DB and DIRECT study
Premenopausal women (<50 years)
Received NAC Excluded:

• no physiological cycle
• no serum in biobank
• serum collected at time point other
than 0–5 days before NAC
Patients included in the analysis
b c d
Progesterone-low 100
AvL-NKI Progesterone-low
100
DIRECT Progesterone-high *
50

Tumour burden
50
Tumour burden
0
Progesterone-high 0
–50
–50
–100 –100
Complete response Progesterone:
T2N1M0
T3N0Mx
T3N0M0
T2N0M0
T1cN1M0
T2-3N1M0
T2N0M0
T2mN1M1
T2N0Mx
T2N0
T3N1M0
T2N0
T2N1
T3N1
T2N3M0
T3N3bM1
T2mN1
T2N1
T2N0M0
T4N2M0
T3N1
T2N0M0
T2N1M0
T2N1M0
T4N3M0
T2-3N3
T2N1
T2N1
T1N1M0
ND
h
w
ig
Partial response
Lo
H
Steady disease
Progressive disease
Patients (n = 30)
e f Progesterone-low g
Progesterone-low
Progesterone-high 0
0
**

Tumour burden
Tumour burden
Progesterone-high –50
–50
–100 –100
Complete response Progesterone:
T2N3bM0
T3N1M0
T3N1M0
T2N2M1
T3N1M0
T2N3M0
T4N3M1
T2N1M1
T2N0M0
T2N0Mx
T2N3M0
T3N3M0
T3N1M0
T1mN2
T3N1
T2N1
T2N1
T2N0
T4N3
T3Nx
T2N1
T3N0
T2N1
ND
T2
h
w
ig
Lo
H
Partial response
Steady disease
Progressive disease
Patients (n = 25)
Fig. 5 | Retrospective analysis of menstrual cycle-dependent sensitivity individual patients is indicated below bars. Dashed lines indicate −30%
to NAC in patients with BC. a, Flowchart of case-selection processes for the and +20%, which represent the thresholds for response and progression in
HR+HER2− BC cohort (cohort 1) and TNBC cohort (cohort2 ), showing every HR+HER2− tumours (c); −30% indicates minimal reduction in tumour size for
selection step. b,e, Evaluation of BC treatment response to NAC initiated a partial response classification in TNBC (f) based on Response Evaluation
during the progesterone-low (top) and progesterone-high phase (bottom) Criteria in Solid Tumours 1.1111 guidelines. d,g, Change in primary tumour size
in patients with HR+HER2− (b) and TNBC (e), based on Response Evaluation following NAC relative to tumour size before treatment, as a percentage in
Criteria in Solid Tumours 1.1111 criteria. c,f, Change in size of target lesion post patients with HR+HER2− BC (d) and TNBC (g). HR+HER2−: n = 17 (progesterone-
treatment compared with pretreatment, as a percentage, in patients with low) and n = 13 (progesterone-high) at first NAC treatment; TNBC: n = 17
HR+HER2− BC (c) and TNBC (f). Blue and red represent progesterone-low and (progesterone-low) and n = 8 (progesterone-high) at first NAC treatment. Data
-high conditions, respectively. Solid bars indicate patients from the Antoni van shown as violin plots; thicker solid lines represent median, thinner solid lines
Leeuwenhoek Hospital-Netherlands Cancer Institute (AvL-NKI), striped bars indicate the 25th and 75th percentiles and dots represent individual patients.
indicate patients from the Dutch Breast Cancer Research Group DIRECT study, Statistical analysis was performed using two-sided Wilcoxon–Mann–Whitney
with data collected at the Clinical Research centre of Leiden University Medical testing. *P < 0.05, **P < 0.01. Further details on statistical analysis are provided
Center (LUMC). Tumour (T), lymph node (N), metastases (M) staging of in Supplementary File 1.
(cohort 1), resulting in a cohort of 25 patients (cohort 2). Progester- increase in age in the progesterone-high group (Extended Data Fig. 6h–l
one level assessments classified 17 patients in the progesterone-low and Extended Data Table 2). In line with the results obtained from mice
phase and eight in the progesterone-high phase during NAC initiation and the HR+HER2− BC group, the outcomes for patients with TNBC
(Extended Data Fig. 6g). Tumour size and grade of this TNBC cohort indicate that NAC initiated during the progesterone-high phase was less
were similar between the two groups, with a marginally significant effective, as evidenced by fewer instances of achieving a pathological
complete response and reduced tumour shrinkage, compared with potential vasoconstriction during the former. Moreover, during dioes-
treatment started in the progesterone-low phase (Fig. 5e–g). Together, trus, our data point to an increase in cells undergoing the EMT process,
these data are in line with our mouse data showing that cycle stage is a which has been extensively linked to chemoresistance2–4. Our findings,
determinant of sensitivity to NAC. although shedding light on specific mechanisms, also underscore the
multifaceted nature of chemotherapy sensitivity, and suggest that the
menstrual cycle phase may, in addition to proliferation differences,
Discussion influence chemosensitivity through a multitude of systemic and local-
Globally, over 30% of women diagnosed with BC are premenopausal48. ized changes.
Even with a more personalized treatment strategy based on the histo- Although the physiology of the female body changes substantially
pathologic classification of BC, the response to NAC is still highly het- during the menstrual cycle, therapies have not been extensively
erogeneous and challenging to predict49. Our investigation highlights adapted to or optimized for these oscillating physiological changes.
the oestrous/menstrual cycle as an important factor influencing this Our data warrant investigation in future clinical studies into the men-
variability in chemosensitivity among patients with DC. One limitation strual cycle as a factor that influences BC outcome. This is especially
worth noting is the sample size for our human retrospective studies. relevant if one considers the ‘life years gained’ that can be obtained
Although this may appear modest, it is essential to understand that the from treatment at premenopausal age. This insight into the interaction
studies in the two human cohorts were not designed to deliver clinical between oestrous/menstrual cycle stages and therapeutic efficacy
conclusions, but acted as a foundational proof of concept, grounded raises broader questions beyond BC: could there be similar interactions
in our findings from three distinct genetic mouse models for BC. in other diseases influenced by hormonal changes or other physiologi-
Most chemotherapeutics target rapidly proliferating cells31 and, in cal rhythms? This exploration might redefine how we approach the
line with this idea, we found the best responses when NAC was initi- treatment of numerous conditions, emphasizing the importance of
ated at the most proliferative stage of the menstrual cycle. However, understanding the body’s natural rhythms and their interplay with
although most cells in mouse MMTV-PyMT and Brca1−/−;Trp53−/− tumours medical interventions. For optimization of therapy response, future
and human TNBC tumours do not express hormone receptors research should delve deeper into the myriad of mechanisms contrib-
(Fig. 3)26,30,50, our data demonstrate that the chemosensitivity of these uting to NAC sensitivity during different oestrous/menstrual cycle
tumours still differs between cycle phases. Clinical studies performed stages. Moreover, prospective clinical trials will be essential to ascertain
in the 1980s hinted at this and showed increased therapy responses whether the timing of NAC in relation to the menstrual cycle stage can
following boosting of BC proliferation by oestrogen administration indeed increase chemotherapeutic efficacy in patients with BC, and
before chemotherapy treatment; however, this observation could not potentially in other types of cancer.
be confirmed in a larger study cohort51,52. This suggests that hormone
receptor-mediated proliferation may not be the only factor determin-
ing chemosensitivity. In line with this, the high sensitivity to NAC in Online content
ovariectomized mice suggests that mice do not have enhanced NAC Any methods, additional references, Nature Portfolio reporting summa-
sensitivity in the proliferative oestrus stage, but rather exhibit reduced ries, source data, extended data, supplementary information, acknowl-
chemosensitivity in the dioestrus stage. What other factors can con- edgements, peer review information; details of author contributions
tribute to this differential sensitivity to NAC? It is important to realize and competing interests; and statements of data and code availability
that, although oestrogen and progesterone may be the local executing are available at https://doi.org/10.1038/s41586-024-08276-1.
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the neoadjuvant GeparQuinto trial. PLoS ONE 8, e79775 (2013). © The Author(s) 2024
Methods For staining and treatment experiments in transplanted models,
tumours from three independent, treatment-naive MMTV-PyMT;
Patient samples MMTV-Cre;R26-LSL-YFP;E-cad-mCFP donors were harvested. Mammary
All retrospective medical data/biospecimen studies at the Netherlands tumours were minced and enzymatically digested by gentle shaking
Cancer Institute have been executed pursuant to Dutch legislation and for 30 min at 37 C in digestion mix (0.2% trypsin from bovine pancreas;
international standards. Before 25 May 2018, national legislation on Sigma) and 0.2% collagenase A (Roche)). The digested tumours were
data protection applied, as well as the International Guideline on Good spun down and cell fragments embedded in basal membrane extract
Clinical Practice. From 25 May 2018 we also adhered to the General Data (BME) (RGF BME type 2 PathClear). Mammary tumour organoid
Protection Regulation of the European Union. Within this framework, medium contained DMEM/F12 GlutaMAX (GIBCO), 2% B27 (Invitrogen)
patients are informed and have always had the opportunity to object and 10 ng ml−1 fibroblast growth factor. Brca1−/−Trp53−/− organoids were
or actively consent to the (continued) use of their personal data and a gift from J. Jonkers. Both MMTV-PyMT and Brca1−/−Trp53−/− organoids
biospecimens in research. Hence, the procedures comply both with were orthotopically transplanted into both fourth mammary glands
(inter)national legislative and ethical standards. All retrospective medi- of 8–12-week-old FVB/NRj female mice ( Janvier Labs).
cal data/biospecimens provided by LUMC were part of the DIRECT For transplantation of MMTV-PyMT organoids, 250,000 single cells
study (NCT02126449 (ref. 45)), were conducted in accordance with were plated 3 days before transplantation. On the day of transplanta-
the Declaration of Helsinki (October 2013) and were approved by the tion, BME was dissolved by mechanical disruption and cells dissolved in
Ethics Committee of LUMC in agreement with Dutch law for medical 100 μl of BME type 2 (RGF BME type 2 PathClear)/PBS. For transplanta-
research involving human subjects. tion of Brca1−/−Trp53−/− organoids, 10.000 single cells were transplanted
Assessment of HER2 status in this study followed established guide- in both fourth mammary glands of FVB/NRj mice.
lines, with scoring based on both immunohistochemistry (IHC) and
in situ hybridization techniques. Immunohistochemistry results Staging of mice
were categorized as 0/1+ (HER2-negative), 2+ (equivocal; considered For determination of the oestrous stage of mice, a vaginal smear was col-
positive if amplification was detected with in situ hybridization) or 3+ lected and analysed as described in refs. 24,46,64. In short, the vagina
(HER2-positive). Assessment of oestrogen receptor was performed was flushed with 50 µl of PBS which was then transferred to a glass slide.
according to Dutch guidelines, with scores of 10% or above consid- Following air drying, the slide was stained with 0.01% crystal violet and
ered positive, and those below 10% negative. Pathological complete oestrus stage determined by examination of cytological characteristics
response (pCR) scoring adhered to local standard guidelines. For one using a light microscope. Mice were categorized as regularly cycling
patient, pCR was determined based on radiological images because sur- when they showed cycles of length 4–6 days37.
gery was not performed due to pCR having been achieved. For measure-
ment of tumour reduction from baseline, we compared pretreatment Ovariectomy
radiological images with post-treatment residual disease as assessed In a subgroup of mice, ovariectomy was performed when mammary
by pathological evaluation. tumours of volume 50 mm3 could be palpated, after which mice were
allowed to recover for at least 7 days before IVM or treatment.
Experimental model and subject data
All mice were adult females, housed under a 12/12 h light/dark cycle and Chemotherapeutic treatment of mice
under specific-pathogen-free laboratory conditions and received food When tumours had reached a cumulative volume of 500–750 mm3
and water ad libitum. All experiments were approved and performed following short-term treatment, or either 250–450 mm3 (transgenic
according to the guidelines of the Animal Welfare Committees of the MMTV-PyMT model) or 50–200 mm3 (transplanted models) for long-
Royal Dutch Academy for Sciences (Hubrecht Institute), the Nether- term treatment, mice were treated with either vehicle (PBS) or chemo-
lands Cancer Institute or KU Leuven. Sample size was not determined therapeutic—either doxorubicin hydrochloride (5 mg kg−1; catalogue
a priori. Mice were randomly assigned to experimental groups. All no. D1515, Sigma-Aldrich) or cyclophosphamide monohydrate
experiments were performed in a blinded manner, except for tumour (100 mg kg−1; catalogue no. C7397, Sigma-Aldrich)—once per week by
volume measurement, which was performed by the same investigator intravenous injection. Treatment cycles were continued for a maxi-
who administered chemotherapy treatment, making it impossible to mum of 5 weeks or until the humane endpoint was reached (cumulative
work in a blinded manner. tumour load over 750 mm3 or single tumour over 350 mm3). Tumour
MMTV-PyMT26 and MMTV-Cre61 mice were purchased from Jack- diameter was measured three times per week by calipers. Following
son Laboratory. E-cad-mCFP mice62 were a gift from H. Clevers, R26- the last treatment cycle, primary tumours and lungs were collected
loxP-stop-loxP-YFP (R26R-YFP) mice a gift from J. Deschamps and from these mice for further analyses.
MMTV-Wnt1 (ref. 30) mice a gift from J. Hilkens. MMTV-PyMT;R26R-
Confetti;R26-CreERT2 mice62,63, of a mixed BL6/FVB genetic back- Depletion experiments
ground, were used to label and trace single cells by IVM. Following When mice approached a cumulative tumour volume of 500–750 mm3,
tumour development, mice were intraperitoneally injected with they began receiving depletion antibodies at least 1 week before chemo-
tamoxifen (Sigma-Aldrich; 1.5 mg 25 g−1, diluted in sunflower oil) to therapeutic treatment and this continued until the end of the experi-
activate Cre recombinase and induce colour randomization of the ment. Individual mice were treated every week with either 60 mg kg−1
confetti cassette. MMTV-Wnt1;R26R-Confetti:R26-CreERT2 mice62,63, of either InVivoMAb anti-mouse CSF1R (CD115) monoclonal antibody
of a mixed BL6/FVB genetic background, were used to isolate tumour (BioXCell, clone AFS98, catalogue no. BE0213) or InVivoMAb rat
pieces, which were transplanted into NOD.Cg-Prkdcscid Il2rgtm- IgG2a isotype control (BioXCell, clone 2A3, catalogue no. BE0089), or
1Wjl/SzJ (NSG) female mice (8–12 weeks of age) Following tumour received InVivoMAb anti-mouse CD4 (BioXCell, clone GK1.5, catalogue
development, mice were intraperitoneally injected with tamoxifen no. BE003-1) and InVivoMAb anti-mouse CD8 (BioXCell, clone YTS 169.4,
(Sigma-Aldrich; 1.5 mg 25 g−1, diluted in sunflower oil) to activate Cre catalogue no. BP0117), three times per week, every other day, with a
recombinase and induce colour randomization of the confetti cas- first dose of 400 µg followed by 200 µg per antibody.
sette. Staining and treatment experiments in transgenic mice were
performed on MMTV-PyMT;MMTV-Cre;R26-LSL-YFP;E-cad-mCFP and Mammary imaging window implantation and IVM
MMTV-Wnt1;MMTV-Cre; R26-LSL-YFP;E-cad-mCFP mice of a pure FVB To follow in vivo tumour clone dynamics, a mammary imaging window
genetic background. was inserted into tumour-bearing mice 2–3 days following induction of
Article
lineage tracing. Mice were anaesthetized using a mixture of 2% isoflu- using either the EnVision+ HRP Labelled Polymer anti-rabbit/mouse
rane and compressed air, with surgery performed under aseptic condi- system (Dako, catalogue nos. K400, K4003 and K4001) or Streptavidin/
tions. Before surgery, the skin overlying the tumour was shaved and HRP (Dako, catalogue no. P0397) and the Liquid DAB+ Substrate Chro-
the skin disinfected using 70% ethanol. An incision was made through mogen System (Dako, catalogue no. K3468); counterstaining with
the skin overlying the tumour and an imaging window inserted65,66. haaematoxylin was then performed. Slides were digitally processed
The imaging window was secured using a non-absorbable, non-woven, using a PANNORAMIC 1000 whole-slide scanner (3DHISTECH) and
purse‐string suture (4‐0 prolene suture) in the skin. For imaging, mice images captured with SlideViewer 2.7 (3DHISTECH). For determina-
were sedated using isoflurane inhalation anaesthesia (roughly 2.0% tion of positive cells, IHC staining was quantified using QuPath 0.4.4
isoflurane/compressed air mixture) and received 200 µl of sterile (GitHub) with atomized classifiers.
PBS by subcutaneous injection to prevent dehydration. Mice were
placed in a custom-designed imaging box on the microscope stage and Immunostaining
maintained under constant anaesthesia under the microscope within Tumour samples were fixed in periodate/lysine/4% paraformaldehyde
a temperature-adjusted climate chamber (34.5 °C). Following each buffer overnight at 4 °C, incubated in 30% sucrose overnight at 4 °C and
imaging session, mice were allowed to recover on a heat mat. Imaging embedded in Tissue Freezing medium (Leica Biosystems). Tumours
was performed on either an inverted Leica TCS SP5 AOBS multiphoton were cryosectioned and immunostaining performed on 10 µm sections.
microscope with a chameleon Ti:Sapphire pumped Optical Parametric For this, sections were hydrated in PBS for 10 min at room temperature
Oscillator (Coherent Inc.; www.coherent.com) or a Leica SP8-DIVE and subsequently blocked and permeabilized for 1 h with 0.5% Tri-
confocal microscope equipped with an Insight X3 dual-beam pulsed tonX/5% normal goat serum (NGS) in PBS. The primary antibody used
layer (Spectra Physics; www.spectra-physics.com), equipped with was anti-PHH3 (Millipore, catalogue no. 06-570, 1:500); the antibody
non-descanned detectors. CFP was excited at a wavelength of 840 nm, was diluted in 0.1% TritonX/5% NGS in PBS, and tissues were sectioned
and yellow fluorescent protein (YFP), green flourescent protein and and stained overnight at 4 °C. Following three washes in PBS, the sec-
red flourescent protein at a wavelength of 960 nm. All images were in ondary antibody (donkey anti-rabbit AF568; Invitrogen, catalogue no.
12-bit and acquired with a ×25 (HCX IRAPO, numerical aperture 0.95, A10042, 1:1,000) was incubated for 1 h at room temperature and nuclei
working distance 2.5 mm) water objective with a free working distance stained with TO-PRO-3 (Invitrogen, catalogue no. T3605, 1:5,000).
of 2.40 mm. Three-dimensional tile scans of large tumour areas were Following three 10 min washes in PBS, stained sections were mounted
taken at the indicated time points (at least 5.0 × 5.0 × 0.5 mm3 (x, y, z)) using VectaShield. All stainings were imaged with an inverted Leica TCS
with z-steps of 5–10 µm. Tile scans were collected at regular intervals SP8 confocal microscope. Fluorophores were excited as follows: AF594
over time periods of up to 5 weeks following induction of lineage trac- at 561 nm, YFP at 514 nm and TO-PRO-3 at 633 nm. YFP was collected at
ing. The same imaging fields were followed during subsequent imaging 519–555 nm, Alexa-568 at 575–630 nm and TO-PRO-3 at 644–694 nm. All
sessions, using the imaging coordinates of the first imaging session. images were collected in 12-bit with a ×25 water-immersion objective
(HC FLUOTAR L, numerical aperture 0.95, working distance 2.4 mm).
Postprocessing and analysis of IVM data For determination of positive cells, staining was quantified using Fiji/
For analysis of three-dimensional tile scans, all intravital images were ImageJ v.1.49k and Excel 2016.
stitched and processed using Leica Application Suite X software (Leica
Microsystems) and ImageJ software (https://imagej.net). Clones were EdU detection
manually annotated in the images, and clone size (number of cells) EdU was injected intraperitoneally 4 h before mice were killed (1 mg
counted in three dimensions throughout the z-stacks. By following the per animal, 5 mg ml−1 stock in PBS; Sigma, catalogue no. 900584). EdU
same clones in our intravital images, we quantified individual clone staining was performed on paraffin sections of thickness 10 µm; sec-
sizes over time. The fraction of clones that behaved in a synchronized tions were deparaffinized, and hot target retrieval performed in citrate
manner with phases of growth, when clones become larger, followed buffer pH 6.0. EdU was detected by incubation with 100 mM Tris pH 8.5,
by a phase of regression during which the clones become smaller, 1 mM CuSO4, 100 mM ascorbic acid and 10 μM AlexaFluor-488 azide
was denoted as alternating. To distinguish synchronized clones from (Invitrogen, catalogue no. A10266) for 30 min at room temperature.
non-synchronized in quantitative terms, we identified as synchronized Subsequently, DNA counterstaining was performed with TO-PRO-3
clones those that showed clear, regular alternating phases of growth (Invitrogen, catalogue no. T3605, 1:5,000) in 0.1% TritonX/5% NGS
and regression with the same periodicity, similar to a sinusoidal wave, in PBS for 1 h at room temperature. Following three 10 min washes in
over multiple consecutive cycles throughout the imaging sessions. By PBS, stained sections were mounted using VectaShield. All staining
contrast, non-synchronized clones did not exhibit this regular pattern. was imaged with an inverted Leica TCS SP8 confocal microscopes.
Fluorophores were excited as follows: AF488 at 488 nm and TO-PRO-3
Histochemistry and immunohistopathology at 633 nm; AF488 was collected at 492–530 nm and TO-PRO-3 at 650–
Mouse tissues were formalin fixed and paraffin embedded. Haema- 700 nm. All images were collected in 12-bit with a ×25 water-immersion
toxylin and eosin staining was performed on 2 µm sections and IHC objective (HC FLUOTAR L numerical aperture 0.95 W, VISIR 0.17, FWD
staining on 4 µm sections using routine procedures. For IHC staining, 2.4 mm). For determination of positive cells, staining was quantified
antigen retrieval was performed with Tris/EDTA (pH 9.0) (Tris: Sigma, using semiautomated macros in Fiji/ImageJ v.1.49k and Excel 2016.
catalogue no. 252859; EDTA: Sigma, catalogue no. EDS) for ER-α (Inv-
itrogen, clone 6F11, catalogue no. MA1-27107, 1:100), CD4 (Invitrogen, Flow-cytometric analysis of dying cells
clone 4SM95, catalogue no. 14-9766-82, 1:1,000), CD8 (Invitrogen, Mammary tumours were collected separately and minced on ice using
clone 4SM15, catalogue no. 14-0808-82, 1:2,000), CD31 (Cell Signal- sterile scalpels, followed by digestion for depletion experiments in
ing, catalogue no. 77699S, 1:100), F4/80 (Cell Signaling, catalogue no. 25 μg ml−1 DNase I (Roche) and 5 Wünsch units TH Liberase ml−1 (Roche)
70076S, 1:1,000) or citrate buffer (pH 6.0) for progesterone receptor in PBS at 37 C for 35 min, or in 25 μg ml−1 DNase I (Roche) and 3 mg ml−1
(Fisher Scientific, clone SP2, catalogue no. RM-9102-S1, 1:300). Sec- collagenase A (Sigma) in PBS at 37 °C for 1 h. The digestion mix was fil-
tions were incubated with primary antibodies overnight at 4 °C. For tered through a 70 µm filter (BD Falcon) while adding DMEM/F12 + Glu-
CD4 and CD8 staining, additional labelling was performed by incuba- taMAX, followed by spinning down for 4 min at 500 relative centrifugal
tion of goat anti-rat biotin (SouthernBiotech, catalogue no. 3052-08, force at 4 °C and resuspension of pellets in 5 mM EDTA/PBS. Cells were
1:150) antibody for 30 min at room temperature. Following washing, washed once in 5 mM EDTA/PBS and centrifuged (4 min at 500 rela-
binding of primary antibody was visualized dependent on species tive centrifugal force at room temperature) before proceeding with
antibody labelling. Tumour cells were blocked in fluorescent activated containing low progesterone levels. Analysis of patient samples (sera)
cell-sorting buffer supplied with 20% normal goat serum (Gibco) for was performed blinded, and linked to tumour volume measurements
10 min on ice, before labelling with one of the following antibody com- and other clinical parameters only at a later stage.
binations for depletion experiments: (1) E-cad-eFluor660 (catalogue
no. DECMA-1, eBioscience, 1:200), biotin-conjugated anti-mouse CD41 Statistics and reproducibility
clone eBioMWReg30 (eBioscience, catalogue no. 13-0411-821, 1:200) Statistical analyses were performed using R v.4.4.2 (R Development
and anti-mouse CD45 clone 30-F11 (eBioscience, catalogue no. 13-0451- Core Team and the R Foundation for Statistical Computing) by inte-
85, 1:200); (2) E-cad-eFluor660 (catalogue no. DECMA-1, eBioscience, gration of software from open-source packages. For further details on
1:200); and (3) anti-mouse CD45-BUV395 clone 30-F11 (BD Bioscience, statistics see Supplementary File 1. The level of statistical significance
catalogue no. 564279, 1:200). Secondary labelling was performed using was set at #P < 0.1, *P < 0.05, **P < 0.01, ***P < 0.001. For all violin plots,
streptavidin-conjugated PerCP (BioLegend, catalogue no. 405213, thicker solid centre lines represent the median, thinner solid lines the
1:200). Dead cells were stained using the Apoptosis detection kit PE 25th and 75th percentiles. Statistical analyses of longitudinal mouse
(Invitrogen, catalogue no. 88-8102-74) according to the manufacturer’s data were performed using R v.4.4.2 (R Development Core Team and
protocol. For depletion experiments no secondary labelling was per- the R Foundation for Statistical Computing) by integrating software
formed and, following washing, cells were immediately stained with from open-source packages, including nlme67, JMbayes2 (ref. 68) and
PI and analysed on a Symphony A5 (BD Biosciences) then washed once packages from tidyverse69, including dplyr, tidyr and ggplot2, and
in 5 nM EDTA/PBS and analysed on a Symphony A5 (BD Biosciences). were analysed as follows. Both tumour volume over time and time to
A broad forward scatter/side scatter (FSC/SSC) gate was followed by event (either end of study or death) were recorded for each individual
gates excluding doublets. Immune cells and megakaryocytes were then animal. To test for survival, survival regression (Cox) models were used
excluded, based on staining for CD41 and CD45, in a dump channel. to test whether survival probability between groups was different. For
Tumour cells were selected according to YFP positivity and further longitudinal tumour measurements, the mixed-effects model70 was
stringently gated for the presence of the cell death marker annexin used. Regression splines (natural cubic splines with 2 or 3 degrees of
and PI, or for PI only for depletion experiments. Data were manually freedom, depending on the case) were used to fit a nonlinear tumour
analysed with FlowJo v.10.6.2. Analysis of staining and fluorescent growth function per animal along time, taking into account all sepa-
activated cell-sorted samples was pseudomized by number coding. rate tumours for each individual animal separately. This model takes
the correlated structure of the data into consideration by inclusion
RNA isolation, complementary DNA preparation and qPCR of individual animals as a grouping factor, and tumour (within the
RNA was isolated using Trizol reagent (Invitrogen Life Technologies) animal) as a subgrouping factor. Furthermore, the effects of time and
according to the manufacturer’s protocol. Purity and amount of RNA treatment were considered as random and fixed effects, respectively.
isolated were analysed using a Nanodrop spectrophotometer. Com- A joint model was used to combine survival and tumour volume meas-
plementary DNA was prepared using the High-Capacity cDNA Reverse urements, to determine whether the responses together were different
Transcription Kit (Applied Biosystems) according to the manufacturer’s between treatment groups. Splines were again used to represent the
protocol. Sequences of used primers can be found below. Quantitative effect of time. For this model, multiple tumours per animal had their
PCR (qPCR) was performed using Power SYBR Green PCR Master Mix growth curves averaged. For further details on these statistics, see
(Applied Biosystems). Thermal cycle conditions used for all qPCR reac- Supplementary File 2.
tions were as follows: 5 min at 95 °C, followed by 40 cycles of denatura-
tion for 30 s at 95 °C, annealing for 30 s at 60 °C and extension for 1 min Reporting summary
at 72 °C. PCR reactions were concluded with incubation for 10 min at Further information on research design is available in the Nature Port-
72 °C to complete the extension of all synthesized products. folio Reporting Summary linked to this article.
Progesterone assay
To 250 µl serum or calibrator samples was added 10 µl of deuterated Data availability
internal standard (progesterone-d9, CDN Isotopes). Subsequently, All statistical analyses are available in Supplementary Files 1 and 2.
progesterone was extracted with 1 ml of methyl tert-butyl ether and Source data are provided with this paper.
dried using a SpeedVac concentrator. Dried samples were reconsti-
tuted in 100 µl of injection solution (methanol:water 2:3, v:v). There-
after, samples were centrifuged for 5.0 min at 18,213g and 50 µl was Code availability
injected into a Nexera SIL30ACMP (Shimadzu) autosampler. Chro- The code generated is available in Supplementary File 1.
matographic separation was achieved using a Kinetex EVO 1.7 µm C18
column (2.1 mm, internal diameter 50 mm) (Phenomenex). A gradient 61. Wagner, K. U. et al. Cre-mediated gene deletion in the mammary gland. Nucleic Acids
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Article
Acknowledgements We thank M. Liefaard and E. Lips for the kind donation of a serum samples at NKI. V.C.M.G. acquired patient data. R.M. and L.A. conceived and designed the
sample from their clinical study. We thank the staff at the NKI animal, animal pathology nonlinear nested mixed model. Using this model, L.A. performed longitudinal analysis of the
and flow-cytometry facilities; and the preclinical intervention unit of the Mouse Clinic for animal experiments. R.M. carried out all general statistical tests as outlined in Supplementary
Cancer and Ageing at NKI for their technical support in performing some of the animal File 1. N.d.G. and J.R.K. collected patient samples and data from the DIRECT study. J.W., S.L.
experiments. This work was supported by Boehringer Ingelheim Foundation (PhD and M.K. interpreted and supervised patient data from NKI. J.v.R. conceived the idea, designed
fellowship); an EMBO postdoctoral fellowship (grant no. ALTF 1035-2020) and the Beug experiments and supervised the project. L.B., C.L.G.J.S. and J.v.R. wrote the manuscript, which
Metastasis Prize to C.L.G.J.S.; a PhD fellowship of the Flanders Research Organization has been reviewed and approved by all authors.
(FWO, grant no. 11O4423N) to M.C.; the Ammodo Science Award; the Doctor Josef Steiner
Foundation; and the Netherlands Organization for Scientific Research (NWO, Gravitation Competing interests The authors declare no competing interests.
programme IMAGINE!, project no. 24.005.009 and VICI project no. 09150182110004) to
J.v.R. This manuscript was edited at Life Science Editors, and edited for typos and style by Additional information
ChatGPT. Supplementary information The online version contains supplementary material available at
https://doi.org/10.1038/s41586-024-08276-1.
Author contributions L.B. and C.L.G.J.S. conceived the idea and designed, performed and Correspondence and requests for materials should be addressed to Colinda L. G. J. Scheele
analysed most of the experiments. L.J.v.W., M.L. and H.H.v.R. designed, performed and or Jacco van Rheenen.
analysed serum progesterone level experiments. B.d.B. performed IVM in ovariectomized Peer review information Nature thanks Wendy Ingman, Nicholas Turner and the other,
mice. N.P., M.B. and M.v.d.V. performed ovariectomy experiments. J.O.L., M.C., G.B. and E.N.D. anonymous, reviewer(s) for their contribution to the peer review of this work.
helped with performing and analysing experiments. A.B. acquired and managed patient Reprints and permissions information is available at http://www.nature.com/reprints.
Extended Data Fig. 1 | Oestrous cycle dependent proliferation of tumour highlight growth of individual clones. Top panels show coordinated clonal
cells during oestrus stage in genetic MMTV-PyMT model. a, Left panel: growth, which is abolished in the ovariectomized conditions (bottom panels).
Schematic of healthy mammary gland turnover during the oestrous cycle d, Images of crystal violet stained vaginal smears in oestrus (left) and dioestrus
(centre) and representative immunofluorescent images (3D rendered) of (right) representative for n > 30 mice. The proportion of epithelial cells and
tumour-free mammary ducts during dioestrus (left) and oestrus (right) stage. leukocytes was used to determine the cycle stage. Scale bars, 100 µm. e, Serum
Right panel: representative immunofluorescent image of tumour-free levels of progesterone in different oestrous cycle stages, determined by
mammary ducts in an ovariectomized mouse. Mammary ducts are labelled with cytology of the vaginal smear. n = 9 (oestrus) and n = 10 (dioestrus) mice.
smooth muscle actin (white). Scale bars, 100 µm. b, Schematic representation f, Representative immunofluorescent images of EdU incorporation in tumours
of R26-CreERT2 and R26R-Confetti constructs, which were crossed with different derived from the genetic MMTV-PyMT mouse model during oestrus and
tumour models to enable lineage tracing in a subset of tumour cells. Upon dioestrus phase. Scale bars, 1 mm. g, Expression of the gene PyMT in different
injection of low dose of tamoxifen, some tumour cells will recombine the oestrous stages depicted as violin plot. n = 10 mice per oestrous stage. Data is
confetti construct leading to stochastic expression of one of the four confetti depicted as violin plots thick solid line represents median and solid lines
colours. These single cells can be followed over multiple days using IVM. indicate the 25th and 75th percentiles (e,g). Dots represent different mice (e)
c, Clone growth rate of all quantified clones from multiday IVM imaging of the and tumours from different mice (g). Statistical analysis was performed using
genetic MMTV-PyMT tumour model in physiologically cycling mice (top) or one-sided (e) and two-sided (g) Wilcoxon-Mann-Whitney test. *P < 0.05. More
ovariectomized mice (bottom). Each line represents a clone, and coloured lines details on statistical analysis can be found in Supplementary File 1.
Article
Extended Data Fig. 2 | Oestrous cycle dependent proliferation of tumour lines highlight coordinated growth of individual clones. c, Immunofluorescent
cells during oestrus stage can be reproduced in the MMTV-Wnt1 tumour images (left) and violin plots depicting quantification (right) of EdU incorporation
model. a, Schematic representation of the multiday intravital microscopy in the genetic MMTV-Wnt1 model representative for n = 5 (oestrus) and n = 4
setup (left) with overview images (single Z-plane) (centre) of the first imaging (dioestrus) mice. Scale bar, 100 µm. Data is depicted as violin plots, thick solid
session (top) and the following imaging session (bottom) that are presentative line represents median and solid lines indicate the 25th and 75th percentiles,
for 6 experiments. Rectangles indicate example clones represented over each dot represents a tumour (c). Statistical analysis was performed using
multiple imaging days (right). Scale bar, 100 µm (overview), 50 µm (zoom). one-sided Wilcoxon-Mann-Whitney test (c). *P < 0.05. More details on
b, Clone growth rate of all quantified clones from multiday IVM imaging of statistical analysis can be found in Supplementary File 1.
transplanted MMTV-Wnt1 model. Each line represents a clone, and coloured
Extended Data Fig. 3 | See next page for caption.
Article
Extended Data Fig. 3 | Disruption of physiological oestrous cycle after or long-term (LT) after treatment at endpoint, oestrus (top) or dioestrus
chemotherapy. a, Representative plots depicting the gating strategy to (bottom). Images are representative for n = 10 mice (oestrus, Day 0), n = 7 mice
determine PI+ tumour cells by flow cytometry analysis. b, Representative (oestrus, Day 7), n = 4 mice (oestrus, LT), n = 10 mice (dioestrus, Day 0), n = 7
examples of crystal violet stained vaginal smears in mice with a disrupted cycle mice (dioestrus, Day 7), n = 8 mice (dioestrus, LT). e, Bar graph depicting the
after treatment with chemotherapy. c, H&E image of physiological murine amount of PrF: Primordial follicles; PF: Primary follicles; SF: Secondary follicles
ovary, indicating the different follicle stages: PrF: Primordial follicle; PF: per mm2 in ovaries of mice, pretreatment, 7 days after initial treatment or at
Primary follicle; SF: Secondary follicle; AF: Antral follicle and CL: Corpus endpoint of long-term (LT) experiment. n = O0 = 10; O7 = 7; OLT = 4; D0 = 10; D7 = 7;
luteum. Images are representative for n = 10 treatment naïve mice per stage. DLT = 8. Scale bars, 100 µm (b), 1 mm (c, overview image, d), 100 µm (c, zoom).
d, H&E images of ovaries from mice pretreatment, 7 days after initial treatment
Extended Data Fig. 4 | Differential response in oestrous cycle stages tumour cells identified by positive staining for propidium iodide (e) and
independent of T cell presence. a,b, Representative immunohistochemistry tumour volume change compared to baseline (f) for mice receiving T cell
staining and quantification of CD4+ T cells (a) and CD8+ T cells (b) in treatment depletion antibodies (α-CD4 + α-CD8) and treated with doxorubicin.
naïve MMTV-PyMT mammary tumours during different stages of the oestrous n = Oa-CD4/8 = 5 mice/20 tumours, Da-CD4/8 = 4 mice/16 tumours. Data in a,b,e,f
cycle. n = 3 mice per oestrous stage and 2 tumours per animal. c, Schematic is depicted as violin plots, thick solid line represents median and solid lines
representation of the experimental setup. Mice were treated with depletion indicate the 25th and 75th percentiles. Dots represent individual tumours
antibodies prior to doxorubicin treatment, which was initiated at a cumulative (a,b,e) or mice (f). Scale bars, 500 µm (a,b,d). Statistical analysis was
tumour mass of 500–750 mm3 in either oestrus (O) or dioestrus (D) stage. performed using linear mixed effects models (e), a two way anova (a,b) and
d, Representative immunohistochemistry staining of CD4+ T cells (top) and a one-sided Wilcoxon-Mann-Whitney test (f). *P < 0.05. More details on
CD8+ T cells (bottom) in 36 tumours of 9 mice receiving T cell depletion statistical analysis can be found in Supplementary File 1.
antibodies (α-CD4 + α-CD8). e,f, Violin plots depicting percentage of dying
Article
Extended Data Fig. 5 | Gating strategy for flow cytometry analysis of depletion experiments. Representative plots depicting the gating strategy to determine
PI+ tumour cells by flow cytometry analysis of depletion experiments.
Extended Data Fig. 6 | Retrospective analysis of menstrual cycle-dependent progesterone level in their sera within five days of initiating NAC. h-l, Table and
sensitivity to neoadjuvant chemotherapy in BC patients. a, Patients comparison of standard clinicopathological parameters of 25 premenopausal
with HR+HER2− BC were divided into the progesterone-low (<2.2 nmol/L) or patients with TNBC Data in c,d,e,i,j is depicted as violin plots, thick solid line
progesterone-high (>2.2 nmol/L) group based on the progesterone level in represents median and solid lines indicate the 25th and 75th percentiles.
their sera within five days of initiating NAC. b-f, Table and comparison of Statistical analysis was performed using two way anova (c,d,e), or two-sided
standard clinicopathological parameters of 30 premenopausal patients Wilcoxon-Mann-Whitney test (i,j). #P < 0.1. More details on statistical analysis
with HR+HER2− BC. g, Patients with TNBC were divided into the progesterone- can be found in Supplementary File 1.
low (<2.2 nmol/L) or progesterone-high (>2.2 nmol/L) group based on the
Article
Extended Data Table 1 | Patient information table for HR+HER2- BC patients included in this study
Extended Data Table 2 | Patient information table for TNBC patients included in this study
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The oestrous cycle stage affects mammary

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Clone size (number of cells)

Clone growth rate

Clone growth rate

Clone growth rate

Clone growth rate

100 100 100 100

10–1 10–1 10–1 10–1

d e f DNA Tumour cells

PHH3+ tumour cells per mm2

change from baseline (%)

PI+ tumour cells (%)

PI+ tumour cells (%)

Measurement of tumour volume –50 –50

Progesterone (nmol l–1)

Progesterone (nmol l–1)

Follicles per mm2

Staging (vaginal smear)

Time of treatment 200 Oestrus Time of treatment

Overall survival (%)

first treatment (days)

Stage Stage Stage

Normalized treatment effect

Tumour volume (mm3)

Overall survival (%)

Brca1–/–Trp53–/– Stage at first treatment: Oestrus Dioestrus

Tumour volume (mm3)

Overall survival (%)

Genetic PyMT Transplanted PyMT

Tumour volume (cm3)

intratumour vessels (Pm)

F4/80+ cells (%)

F4/80+ cells (%)

Measurement of tumour volume

change from baseline (%)

Stage Stage Stage Stage

Premenopausal women (<50 years)

Received NAC Excluded:

change from baseline (%)

change from baseline (%)

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