Spatial Proximity to Fibroblasts Impacts Molecular Features and Therapeutic Sensitivity of Breast Cancer Cells Influencing Clinical Outcomes

Abstract

Using a three-dimensional coculture model, we identified significant subtype-specific changes in gene expression, metabolic, and therapeutic sensitivity profiles of breast cancer cells in contact with cancer-associated fibroblasts (CAF). CAF-induced gene expression signatures predicted clinical outcome and immune-related differences in the microenvironment. We found that fibroblasts strongly protect carcinoma cells from lapatinib, attributable to its reduced accumulation in carcinoma cells and an elevated apoptotic threshold. Fibroblasts from normal breast tissues and stromal cultures of brain metastases of breast cancer had similar effects as CAFs. Using synthetic lethality approaches, we identified molecular pathways whose inhibition sensitizes HER2⁺ breast cancer cells to lapatinib both in vitro and in vivo, including JAK2/STAT3 and hyaluronic acid. Neoadjuvant lapatinib therapy in HER2⁺ breast tumors lead to a significant increase of phospho-STAT3⁺ cancer cells and a decrease in the spatial proximity of proliferating (Ki67⁺) cells to CAFs impacting therapeutic responses. Our studies identify CAF-induced physiologically and clinically relevant changes in cancer cells and offer novel approaches for overcoming microenvironment-mediated therapeutic resistance. Cancer Res; 76(22); 6495-506. ©2016 AACR.

Figure 1

Phenotypic changes induced by CAFs. A, Vital immunofluorescence images of 3D Matrigel cultures. Breast carcinoma cells and CAFs are labeled with mCherry (red) and GFP (green), respectively. Scale bars represent 100μm. B, Experimental outline Co-cultured cells are compared to separately cultured carcinoma cells and CAFs that are mixed prior to analysis. C, Volcano plots of CAF-induced changes in gene expression. Statistically significantly different genes up- or down-regulated ≥2 fold are indicated by red and blue, respectively, with corresponding percentages. D, Heat map and hierarchical clustering of expression values of genes that are differentially expressed in at least one cell line. E, Principle component analysis of gene expression profiles Lighter and darker shades denote separate and co-cultures, respectively. Arrows indicate change in profiles. F, Molecular pathways significantly affected by interaction with CAFs, cutoff p<10^-6. G, Overall survival in the METABRIC patient cohort stratified by the T47D signature in Luminal B patients and a combined MCF10DCIS/SUM149 signature in Basal patients. H, Immune and stromal ESTIMATE scores for the corresponding good and poor prognosis patient groups.

Figure 2

Fibroblasts protect breast carcinoma cells from lapatinib. A, Experimental outline. Carcinoma cells expressing luciferase are plated with and without fibroblasts. Following overnight incubation, the cultures are treated with a therapeutic agent Luciferase activity is used as a proxy for viable cell numbers. B, Survival of the indicated carcinoma cells in monocultures and CAFs co-cultures with various concentrations of lapatinib. Asterisks indicate statistical significance (* p<005, ** p<001, *** p<0001). C, Protective effect of the indicated stroma on survival of lapatinib-treated MDA-MB-453 cells CAFs – primary breast cancer-associated fibroblasts, NBF – normal breast fibroblasts, NHA- normal human astrocytes, BMS – breast cancer brain metastasis stroma. D, Long-term clonogenic survival of MDA-MB-453 cells following 3 days of lapatinib treatment CAFs were eliminated by puromycin selection and purity of the epithelial cells was confirmed by microscopy. E, Representative images of BrdU⁺ (green) and α-SMA (red) staining in xenografts treated with vehicle or lapatinib Scale bars correspond to 25μm. F, Distribution of distances between BrdU⁺ carcinoma cells and nearest CAFs in xenografts treated with vehicle or lapatinib P-values indicate statistical significance (t-test). Right panel depicts a representative picture with distances indicated by arrows. G, Representative images of Ki67⁺ (green) and α-SMA (red) staining of primary breast tumor samples before and after four weeks of neoadjuvant lapatinib treatment. Scale bars correspond to 25μm. H, Distribution of distances between Ki67⁺ carcinoma cells and nearest CAFs in breast tumors before and after neoadjuvant lapatinib treatment. P-value indicates statistical significance (two-sided t-test).

Figure 3. Mechanisms of stroma-induced lapatinib resistance

A, Accumulation of lapatinib in MDA-MB-453 cells grown in monocultures and CAFs co-cultures assessed by FACS based on lapatinib autofluorescence. B, Immunoblot analysis of the indicated proteins in HCC1954 and CAFs cultured separately or in post-harvest mixtures, compared to co-cultures. C, Impact of CAFs co-cultures on the ability of indicated pro-apoptotic peptides to induce apoptosis in the indicated cell lines Asterisks indicate results of pairwise t test. D, Sensitization to cytotoxic effect of lapatinib by BCL2/BCL-xl inhibitor ABT737. Asterisks indicate p-values of interaction term by 2-way ANOVA* p<005; ** p<001, *** p<0001.

Figure 4

Hyaluronidase sensitizes CAFs to lapatinib. A, The effect of collagenase and hyaluronidase treatment on the morphology of organoids in co-cultures of MDA-MB-453 cells (mCherry) and CAFs (GFP). Scale bars correspond to 100μm. B-D, Impact of collagenase (B), FAK inhibitor (C) and hyaluronidase (D) treatment on lapatinib sensitivity of MD-MB-453 cells in CAF co-cultures. Asterisks indicate p-values of interaction term in 2-way ANOVA. E, Correlation between production of hyaluronan and protection of MDA-MB-453 cells against 20 μM lapatinib among different fibroblasts. F, Sensitization of CAFs toward lapatinib by hyaluronidase treatment p<0001, interaction term in 2-way ANOVA. G, Tumor weights and H, percentage of cells positive for cleaved caspase-3 staining of xenografts treated with hyaluronidase, lapatinib or both. Treatment was started 10 days post injection and continued for 21 days, at which point tumors were harvested. Asterisks indicate statistical significance (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; unpaired Mann-Whitney test). I, Representative images of cleaved caspase-3 staining. Arrows indicate apoptotic cells. Scale bars correspond to 100μm.

Figure 5. Overcoming CAF-mediated resistance

A, Heat map of fold differences between observed and expected cytotoxicity of combinations of lapatinib with the indicated inhibitors. B, Sensitization to lapatinib by JAK inhibitor BSK805 in the indicated cell lines. Asterisks indicate p-values of interaction term in 2-way ANOVA. C, Growth of the indicated xenografts treated with lapatinib (100 mg/kg), BSK805 (50 mg/kg), or both compounds starting on day 18 after injection. Right panels display volumes of individual tumors at day 40. Asterisks indicate the p-values of statistical significance (* p<005; ** p<001, *** p<0001, Mann Whitney test). D, Representative images of pSTAT3⁺ (cyan) and α-SMA (red) staining of primary breast tumor samples before and after neoadjuvant lapatinib treatment. Scale bars correspond to 25μm. E, Bar graph depicting frequency of pSTAT3⁺ breast tumor cells before and after neoadjuvant lapatinib therapy in all samples combined. P value indicates statistical significance by multiple comparisons (1-way ANOVA). F, Heatmap of differences in pSTAT3, Ki67 and SMA in patients before and after neoadjuvant lapatinib treatment. Hierarchical clustering of patient samples into two groups shows a trend between changes in molecular markers with reduction in tumor size (p=01, Wilcoxon test).