Ejecución

Using the aforementioned second strategy (Fig. 13A), a first set of experiments with non- luciferase-labeled TNBC cells (TCs) was performed. In this set of experiments, tumor resection was carried out 30 days after cell injection (engraftment) into the mammary fat pad.

Examination of primary tumor size after mastectomy revealed that sCAV1high tumors (TCs +

CAFscr) were significantly bigger (Fig. 14B and 14D) and showed a clear reddish pigmentation

(Fig. 14A and 14C), as compared with either the sCAV1low group (TCs + CAFshCAV1) or tumors generated by the injection of only cancer cells (TCs). While MDA-MB-436 cells were able to generate a visible tumor when injected alone, MDA-MB-468 cells were less penetrant,

giving rise to tumor masses only in some mice (Fig. 14C).

Figure 14. Stromal CAV1 levels modulate primary tumor growth. (A) Orthotopic xenograft tumors were generated by injecting MDA-MB-436 TCs alone or in combination with CAFs (expressing high or low CAV1 levels, CAFscr

and CAFshCAV1

respectively). Tumors were isolated at 30 days post-cell injection. Representative tumors are shown. Scale bar: 10mm (B) Tumor size was calculated at 30 days post-injection of TC and CAF cells. Data are presented as boxplots (Min to Max values); n=4 mice/group, **P<0.01, ***P<0.001 (One-way ANOVA, Bonferroni’s multiple comparison test). (C) Orthotopic xenograft tumors from isolated MDA-MB-468 breast tumor cells (TCs) or in combination with CAFs. Mice were mastectomized and tumors isolated at 30 days following injection into mammary glands. Representative tumors are shown. Of note, at this time point, no tumor was developed from MDA-MB-468 TCs when injected alone (image depicts mammary fat pads). Scale bar: 10mm. (D) Tumor size was calculated at 30 days post-injection of TC and CAF cells. Data are presented as boxplots (Min to Max values); n=6 mice/group, **P<0.01, (Two-tailed unpaired t-test). (E and F) Tumor size was calculated at 40 days post-injection of breast tumor cells (MDA-MB-436 or MDA- MB-468, respectively) and CAF cells. Difference in volume between CAFscr and CAFshCAV1 derived tumor xenografts is no longer significant although tendency remains. Data are presented as boxplots

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(Min to Max values) and are representative of 2 independent experiments; n=4 mice/group, ***P<0.001 (One-way ANOVA, Bonferroni’s multiple comparison test). (G) RNASeq heatmap of tumor xenograft lysates showing significant changes in expression levels of glycolytic enzymes. Mastectomy was performed 30 days post-cell injection. In gene expression heatmap, red denotes increased gene expression while green corresponds to downregulation. LogFC (Fold Change) of gene expression compared to the MDA-MB-436 + CAFscr control. n=3 tumor/group.

By extending the period of primary tumor growth before resection from the usual 30 to 40 days

MDA-MB-468 cells generated small tumors on their own (Fig. 14F). Upon extension of primary

tumor growth experiments, statistical significance for the difference in size among high and low stromal CAV1 tumors was lost, although trends agreed with the previous significant differences

(Fig. 14E and 14F).

Low stromal CAV1 (sCAV1low) MDA-MB-436 xenografts resected 30 days post-cell injection also showed a significant upregulation of glycolysis-related genes upon RNASeq transcription

analysis of whole tumor xenograft lysates (Fig. 14G and Table S6A).

Analogous experiments were performed using a luciferase-expressing MDA-MB-436 cell line (MDA-MB-436luc_{) to track cancer cell growth non-invasively at different time points (Fig. 15A).}

Again, the presence of CAFs encouraged primary cancer cell growth (Fig. 15B).

Figure 15. Tracking in

vivo tumor cell

growth. (A)

Bioluminescent imaging of mice transplanted with a luciferase-expressing breast cancer cell line (MDA-MB-436luc_{) either}

isolated or in combination with

CAFs. Images

correspond to 23 days post-cell injection time point. Color scale shows luminescence count production. (B) Quantification of tumor cell-derived bioluminescence during primary tumor xenograft growth. Last two data points are not shown as they are not representative of tumor size due to necrosis and tumor tissue opacity. Data are presented as mean ± SEM and are representative of 3

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independent experiments; n=4 mice/group, *P<0.05 compared to MDA-MB-436 luc_{, (Individual time point}

analysis: One-way ANOVA, Tukey’s multiple comparison test).

An intriguing observation was that sCAV1low tumors showed a two-stage growth curve. During the first stage of tumor growth, TCsluc _{from sCAV1low tumors grew faster (Fig. 15B). However,} around day 10 post-cell injection in the mammary fat pad, this behavior was reversed and

TCsluc _{growth in sCAV1low tumors (red hue data) was stalled compared to sCAV1high tumors}

(blue hue data), which continued growing exponentially beyond that time threshold (Fig. 15B).

Supporting an underlying differential proliferation rate for such differences in the later phase, sCAV1high tumors exhibited increased nuclear Ki67-positive cell counts (a standard marker of cell proliferation) upon immunostaining of histological tumor sections (Fig. 16A and 16B).

Figure 16. Proliferation assessment by Ki67 IHC. (A) Ki67 (proliferation marker, in brown) IHC of MDA-MB-436 derived primary breast xenografts removed 30 days post-cell injection. Total cell nuclei are shown upon hematoxylin staining (light blue). Automated image analysis (lower panels) allowed for classification of Ki67+ proliferating cell nuclei (red) and Ki67 – non-proliferating cell nuclei (blue). Scale bar: 1mm. (B) Proliferating (Ki67+) cell quantification in tumor xenograft IHC sections (see Figure S1I). Data are presented as boxplots (Min to Max values); n=4 tumor/group, *P<0.05, ****P<0.0001, (One- way ANOVA, Bonferroni’s multiple comparison test).

In order to isolate these effects, a series of in vitro experiments, where TCs co-cultured together with CAFs of either genotype and then profiled using automated high-content image

analysis were performed (Fig. 17A). In accordance with the first stage of the biphasic tumor

growth observed in vivo, these studies showed that cancer cells co-cultured with CAFshCAV1 fibroblasts multiplied faster than cancer cells cultured alone or in combination with CAFscr

fibroblasts (Fig. 17B and 17C). Quantification of CAFs present in the co-culture assay showed

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Figure 17. In vitro co- culture assay of breast CAFs and TNBC cell lines (TCs) (A) Co-culture assay of CAFs and TCs, showing an input image of GFP positive CAFs (green), total nuclei (blue) and Ki67 staining (red). Presence or absence of GFP allowed for classification of cells into class CAFs (green nuclei) or class TCs (red nuclei), respectively, for later quantification. Scale bar: 20µm. (B and C) Breast tumor cell (TCs) class quantification in MDA-MB- 436 + CAFs and MDA- MB-468 + CAFs co- cultures, respectively, along 7 days. Presence of CAFshCAV1 encourages increased TCs numbers. Note the stalled cell growth in the case of isolated MDA-MB-468 cell line grown in absence of CAFs. Data are presented as mean ± SEM and are representative of 3 independent experiments; n=12 well/group, *P<0.05, ***P<0.001, ****P<0.0001, compared to TCs + CAFscr at Day 6 (Two-way ANOVA, Bonferroni’s multiple comparison test). (D and E) Fibroblast (CAFs) class quantification in MDA-MB-436 and MDA-MB-468 co-cultures, respectively, along 7 days. Cell growth shows no difference among CAFscr and CAFshCAV1 fibroblasts. Note the absence of false positive classifications of fibroblasts in images derived from cancer cell-only (TCs) conditions (in grey). Data are presented as mean ± SEM and are representative of 3 independent experiments; n=12 well/group (Bonferroni’s and Tukey’s multiple comparison test).

Cancer-Associated Fibroblasts (CAFs) had already been described as cancer cell growth promoters45,75–77. With these results, it could be observed how CAV1 levels in stromal fibroblasts dynamically affect the different stages of tumor growth. While CAFshCAV1 fibroblasts promote a quick initial cancer cell proliferation in vitro and in vivo, long-term growth is better supported by CAFscr-containing sCAV1high tumors.

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3.3 Aberrant proangiogenic signaling generates a dysfunctional tumor vasculature in