Identification of Luminal Breast Cancers that Establish a Tumor Supportive Macroenvironment Defined by Pro-Angiogenic Platelets and Bone Marrow Derived Cells

Abstract

Breast cancer recurrence rates vary following treatment, suggesting that tumor cells disseminate early from primary sites but remain indolent indefinitely before progressing to symptomatic disease. The reasons why some indolent disseminated tumors erupt into overt disease are unknown. We discovered a novel process by which certain luminal breast cancer cells and patient tumor specimens (LBC “instigators”) establish a systemic macroenvironment that supports outgrowth of otherwise-indolent disseminated tumors (“responders”). Instigating LBCs secrete cytokines that are absorbed by platelets, which are recruited to responding tumor sites where they aid vessel formation. Instigator-activated bone marrow cells (BMCs) enrich responding tumor cell expression of CD24, an adhesion molecule for platelets, and provide a source of VEGFR2+ tumor vessel cells. This cascade results in growth of responder adenocarcinomas and is abolished when platelet activation is inhibited by aspirin. These findings highlight the macroenvironment as an important component of disease progression that can be exploited therapeutically.

INTRODUCTION

Breast cancer is a heterogeneous disease that is categorized into molecular and histopathological subtypes based predominantly on analysis of hormone and growth factor receptors – namely estrogen (ER), progesterone (PR), and HER2/Erbb2 (Her2) (1). Women with triple-negative breast cancer (TNBC; i.e. ER-/PR-/Her2-) are at the greatest risk of early recurrence (2). Luminal breast cancers (LBC), which often include ER+ tumors, are the most prevalent form of breast cancer. These tumors are often differentiated and associated with good prognosis, yet some patients with LBC experience recurrent disease even 15-20 years after their initial diagnosis and surgery (3). Although classification into these categories has some correlation with patient outcome, it is difficult to accurately predict which patients will relapse. Furthermore, there is no correlation between molecular classification and patient response to current treatment therapies (4).

In some patients with metastatic breast cancer, tumor cells clearly disseminate prior to surgery, but remain undetected for protracted periods of time before the patient becomes symptomatic (5). Incipient primary tumors and second primary tumors can also exist in a state of indolence before being detected. For example, autopsy studies of people without a medical history of cancer revealed that indolent cancers are highly prevalent within the general population (6). What causes indolent tumors to erupt into overt disease is unknown, making it difficult to predict which cancer patients are likely to relapse or to benefit from preemptive therapy.

The systemic environment is appreciated as an important determinant of tumor malignancy and progression (7). We previously established that indolent cancer cells (“responders”) that are disseminated to various anatomical locations within host mice can be stimulated to form malignant tumors as a consequence of aggressively growing triple-negative breast tumors (“instigators”) located at distant anatomical sites (8,9). A growing body of evidence supports the notion that tumors that co-exist within a patient who has multiple tumor burden (e.g., multiple disseminated metastases) can interact systemically to modulate overall cancer progression (10). Responding tumor outgrowth occurs as a consequence of systemically-acting cytokines and bone marrow derived cells that are rendered pro-tumorigenic by the instigating triple-negative breast tumors. This cascade of events, termed “systemic instigation”, results in the outgrowth of highly desmoplastic, malignant tumors (8). We designed studies to determine if other breast cancer subtypes employ these same mechanisms. A deeper understanding of systemic tumor-promoting processes should improve identification of patients who would benefit from adjuvant therapy.

RESULTS

Breast Cancer Subtype Determines Disseminated Tumor Phenotype

In order to understand whether luminal breast cancers (LBC) exert similar protumorigenic systemic effects as instigating triple-negative breast cancers (TNBC), we injected responding human breast cancer HMLER-HR cells (9,11) contralaterally to either LBC tumor cells (MCF7Ras (12)), TNBC tumor cells (BPLER (13)), or Matrigel vehicle control in Nude mice, according to our human tumor xenograft protocol (Fig. 1A). We recovered tissue from 3 of the 4 mice that had been injected with responding tumor cells opposite Matrigel control; however, microscopic analysis revealed that only one tissue plug contained a small responding tumor (~10 mg) while the other two did not form bone fide tumors (Fig. 1B). Responding cells formed tumors in 100% of the mice bearing the systemic environments established by TNBC and LBC (Fig. 1B). Importantly, the resulting responding tumors were formed by the human responder cells that had been injected (Fig. S1A).

Figure 1.

Breast cancer subtype-specific systemic environments affect bone marrow derived cells and phenotype of otherwise indolent tumors at distant sites. A, Systemic instigation human tumor xenograft model. Aggressively growing “Instigating” human tumors or controls are injected into one site of host Nude mice; otherwise indolent “Responding” human tumor cells injected into distant anatomical locations. B, Mass of responding tumors that formed in the systemic environments established by control Matrigel, triple-negative breast cancer (TNBC), or luminal breast cancer (LBC). Incidence of responding tumor formation is indicated above each bar; n=4 mice per group. C, Hematoxylin and eosin (H&E) stains of responding tumors after growth in indicated systemic environments; scale bar = 200 μm; inset = 100 μm. D, Staining for the proliferation marker, Ki67 (brown) in indicated responding tumors; nuclei counterstained with hematoxylin. Areas of necrosis (“N”) and edema (“E”) are indicated. Scale bar = 100 μm. E, Average number of vessels per area in indicated tumors and tissues. Vessels were counted under 40x magnification in 3 different areas from each of 3 different tumors per group (n=9 images per group). The two tissue plugs recovered opposite Matrigel that did not contain focal tumors were also counted. F, Serial sections of LBC-instigated responding tumors stained for CD31-positive endothelial cells (red, left), αSMA-positive pericytes (red, center), and VEGFR2-positive endothelial progenitor cells (red, right). Nuclei counterstained with DAPI (blue). Arrowheads indicate blood vessels. Top row scale bar = 100μm; bottom row represents high magnification of images in top row. G, Whole mount fluorescent images (4x) to visualize GFP+ BMCs recruited to responding tumors after 4 weeks of exposure to indicated systemic environments. Numbers indicate average percentage of total tissue cells that were comprised of GFP+ BMCs; n=4 per group. H, Results from flow cytometric analysis of GFP+ bone marrow-derived cells recruited into responding tumors in (G); n=4 per group. I, Flow cytometric analysis of indicated cells in the marrow of mice bearing Matrigel control or instigating LBC. Graph represents average fold change in numbers of indicated cell types in bone marrow of mice bearing LBC relative to those bearing Matrigel control; n = 4 mice per group. Also see Figs. S1, S2, S3 and S5.

Responding tumors that were instigated by TNBC displayed no observable necrosis and were moderately mitotic (Fig. 1C, D and Fig. S1B, C). As we observed previously (8), these responder tumors formed a desmoplastic stroma infiltrated by alpha-smooth muscle actin (αSMA)-positive myofibroblasts (Fig. S1D). In contrast, responding tumors growing in the LBC environment had areas of observable edema and necrosis, and were highly mitotic (Fig. 1C, D and Fig. S1B, C). These responding tumors were extensively vascularized without forming desmoplastic stroma (Fig. 1C and Fig. S1D). These histopathological phenotypes were consistent with breast adenocarcinomas observed in the clinic (2). The control tumor recovered from the Matrigel environment was comprised of viable responder cells only at the tumor periphery (Fig. 1C,D and Fig. S1B-D).

The differences in responding tumor histopathology suggested that LBC might employ different systemic tumor-promoting mechanisms than TNBC. Indeed, levels of the cytokine osteopontin (OPN), an endocrine factor that is necessary for TNBC-dependent systemic instigation (9) were secreted at ~450-fold lower levels from the LBC tumor cells than from the TNBC tumor cells (p=0.002) and were no different than that of the responding tumor cells (Fig. S1E).

VEGFR2-Positive Cells Incorporate into Vasculature in LBC-Instigated Tumors

Tumors that responded to the TNBC environment had significantly higher vessel density than the control tissues (~2.6-fold); however, those in the LBC environment had higher microvessel density than either control tissues (5.7-fold, p=0.001) or TNBC-induced tumors (2.2-fold, p=0.014) (Figs. 1E and S5A-C). Blood vessels in the LBC-induced responding tumors contained few CD31-positive cells, were weakly positive for mouse endothelial cell antigen (Meca32), and lacked pericyte coverage, as indicated by the absence of associated αSMA-positive cells (Figs. 1F and S2A) even though peri-tumoral vasculature stained strongly for MECA32 and αSMA (Fig. S2A, B). Therefore, we examined responding tumors for the presence of vascular endothelial growth factor receptor 2 (VEGFR2)-positive cells, which aid the formation of blood vessels to varying extents under different pathological conditions (14). In the tumors that responded to LBC-dependent systemic instigation, the vast majority of blood vessels were comprised of VEGFR2+ cells (Fig. 1F). Vasculature in the tumors instigated by TNBC was predominantly devoid of VEGFR2+ cells (Fig. S2C).

VEGFR2+ endothelial precursor cells have been shown to originate in the bone marrow (14,15) and their elevated numbers in the circulation correlate with advanced stage in patients with invasive breast cancer (16). Therefore, we examined recruitment of bone marrow derived cells (BMDCs) into the various responding tumors in Nude mice that had been successfully engrafted with GFP+ BMCs prepared from eGFPRag1^-/- mice (Fig. S3A). We recovered tissue plugs 4 weeks after injection of the responding tumor cells, when the average tissue mass in each group was 10mg (not shown). In the plugs extracted from sites where responding tumor cells had been injected contralaterally to Matrigel, only ~5% of the total cellular portion of these tissues was comprised of GFP+ BMDCs (Fig. 1G). The numbers of BMDCs in these tissues were not significantly different from those of the contralateral Matrigel plug or control lung tissues, which contained ~3% GFP+ cells (Fig. S3B and data not shown). In contrast, BMDCs were incorporated to a significantly greater extent into responder tumors promoted by both TNBC (p=0.006) and LBC (p=0.012) instigators; ~20% of the total cellular portion of these tumors was comprised of GFP+ bone marrow-derived cells (Fig. 1G).

In responding tumors from the LBC environment, VEGFR2+ cells comprised ~50% of the total number of GFP-positive BMDCs (Fig. 1H). These numbers represented a 39% increase above those of TNBC-induced tumors and a 67% increase above tissues in the Matrigel environment. In consonance with our earlier report (8), pro-tumorigenic Sca1+/cKit- hematopoietic BMDCs were incorporated to a significantly greater extent into responding tumors stimulated by TNBC than those recovered opposite Matrigel (Fig. 1H). The contributions of CD11b+/CD45+, Sca+/cKit+, VEGFR1+, and CD31+ cells to the total GFP+ BMDC population were not significantly different between the cohorts of mice (Fig. 1H).

VEGFR2+ cells were also ~2.7-fold more abundant in the bone marrow of mice bearing LBC tumors than in those bearing TNBCs or Matrigel control (Fig. 1I). The numbers of other BMC populations in the marrow, such as CD11b+/CD45+ myeloid cells, were not statistically different between groups (Fig. 1I).

Collectively, these data indicated that luminal breast tumors mediated the expansion of VEGFR2+ bone marrow cells that were subsequently mobilized to distant responding tumor sites. Within the responding tumor microenvironment VEGFR2+ cells contributed, at least in part, to the formation of tumor vasculature.

LBC Enhances Platelet Recruitment to Responding Tumors

We analyzed the plasma levels of some common pro- and anti-angiogenic circulating cytokines as potential mediators of systemic instigation in the LBC environment but found no statistically significant differences between cohorts (data not shown). Others have shown that proteins, including angiogenic regulators, are enriched several hundred-fold in circulating platelets as compared to the plasma and that platelets are potent mediators of angiogenesis (17-19). We therefore reasoned that platelets were ideal candidates as mediators of systemic instigation.

Responding tumors that formed as a consequence of LBC instigation recruited ~3.7-fold more platelets than those stimulated by TNBC (Fig. 2A). We therefore analyzed responding tumors for expression of ligands that are known to mediate platelet adhesion, including Collagen IV (ColIV) and CD24. ColIV is a potent chemoattractant that recruits platelets to injured vessels during wound healing or tumor formation (20). CD24 is a cell surface glycoprotein that is used as a surrogate marker for differentiation status of breast and other cancer cells (21) and can bind to the platelet-expressed adhesion molecule, p-selectin (22).

Figure 2.

Platelets are recruited to responding tumors during LBC systemic instigation. A, Left: Immunofluorescent images of responding tumors in indicated macroenvironments stained for p-selectin to visualize platelets (red); nuclei counterstained with DAPI (blue). Scale bar = 100μm. Right: CellProfiler software outlines of p-selectin-positive areas used for quantification. Graph represents average p-selectin-positive area per image; TNBC n=15 images; LBC n=19 images. B, Masson's Trichrome to visualize collagen (blue), red blood cells (bright red), and cell nuclei (dark brown). Blood vessels (“BV”), and necrotic areas (“N”) are indicated. Scale bar = 100μm. C, CD24 staining (green) of responding tumors under indicated conditions; nuclei counterstained with DAPI (blue); scale bar = 100μm. D, Merged immunofluorescent images of responding tumors or cell plugs that had formed under indicated conditions. Left: CD24 (green), p-selectin (purple). Right: collagen IV (red), p-selectin (purple). BV denotes blood vessel. In all cases, cell nuclei counterstained with DAPI (blue); scale bars = 20μm.

The vast majority of blood vessels within the responding tumors instigated by LBC contained areas of exposed collagen, whereas collagen deposition was predominantly confined to the intra-tumoral extracellular matrix in responders from the matrigel and TNBC environments (Fig. 2B). Moreover, carcinoma cells from responding tumors that had been instigated by LBC were highly enriched for cell surface expression of CD24, particularly in areas surrounding blood vessels (Fig. S3C), when compared with those injected opposite TNBC or Matrigel control, in which CD24 expression was limited to a few responding cells (Fig. 2C). We observed platelets within these CD24-and ColIV-rich areas of LBC-instigated responding tumors compared to similar areas in control responding tumors in which p-selectin-positive platelets were not obvious (Fig. 2D), even though circulating platelet counts were elevated in both cohorts relative to cancer-free mice (Fig. S3D).

These results indicated that different tumor-promoting systemic environments had a profound impact on the ability of tumor cells to recruit platelets.

Platelets are Rendered Pro-Angiogenic by Instigating LBC Tumors

The fact that platelets had selectively accumulated in responding tumors exposed to the LBC systemic environment suggested that they might play a functional role in tumor promotion. Hence, we analyzed the ability of platelets, prepared from various tumor-bearing mice, to stimulate angiogenesis using a standard in vitro human umbilical vein endothelial cell (HUVEC) assay. Resting platelets harvested from cancer-free mice and from mice bearing responding tumors opposite either Matrigel control or LBC tumors induced capillary tube formation to similar extents (Fig. 3A). ADP-activated platelets from mice bearing responding tumors opposite Matrigel control were unable to stimulate angiogenesis above baseline controls (Fig. 3A). In significant contrast, ADP-activated platelets from mice bearing LBC tumors had ~4-fold enhanced angiogenesis-promoting ability (Fig. 3A).

Figure 3.

Platelets in the LBC environment are enhanced for pro-angiogenic function and take up pro-angiogenic factors secreted by LBC tumor cells. A, Left: representative images of capillary tubes formed by human umbilical vein endothelial cells (HUVECs) after 6 hr exposure to platelet releasates prepared from indicated mice; 4x magnification. Right: Quantification of HUVEC branch points over a 4-7 hour time course (see Methods) induced by platelet releasates from indicated tumor-bearing mice. Mouse and platelet status indicated below graph; n=3 samples per group, tested in duplicate. B, Left: representative 4x images of capillary tubes formed by HUVECs after 7 hr exposure to 48-hr conditioned medium (CM) from indicated cell lines. Right: Quantification of HUVEC branch points over a 4-7 hour time course (see Methods) induced by CM from indicated cell lines. Releasates from resting mouse or human platelets were used as controls. All samples analyzed in duplicate. C, Assay to test ability of platelets to absorb pro-angiogenic factors from CM of indicated cell lines. 48-hour CM was collected from LBC instigator cells or responder cells and exposed to naïve platelets from cancer-free humans or mice for 10 min at 37°C. Various media (1A-2B) were tested for ability to induce angiogenesis in the HUVEC assay. D, Relative number of capillary tube branch points induced by CM from (C). HUVECs were subjected in vitro to indicated CM and the number of branch points quantified during a 4-7 hour time course. Data represent relative number of branch points: 1B/1A for resting naïve mouse and human platelets and 2A/2B for resting naïve mouse platelets. All samples were tested in duplicate. E, Relative levels of indicated cytokines in platelet lysates from mice bearing Matrigel or LBC tumors; n=3 mice per group. Significant values: GRO (p=0.012), IFNγ (p=0.050), IL6 (p=0.044), PDGF-BB (p=0.033), and PlGF (p=0.044). F, Instigating or Responding tumors under indicated conditions stained for phosho-STAT3 (red), SV40 LgTAg-positive responder cells (green), and cell nuclei (DAPI, blue); scale bar = 100μm. Also see Fig. S4.

In light of this striking result, we wished to know if the platelet pro-angiogenic potential was directly imparted by instigating LBC tumors. First, we assessed the pro-angiogenic capacity of conditioned medium (CM) from cultured cells. As controls, we used medium from resting mouse and human platelets prepared from cancer-free subjects, which minimally promoted capillary tube formation (Fig. 3B). The CM from responding tumor cells did not significantly enhance in vitro angiogenesis to any extent above that of the control CM from resting platelets (Fig. 3B). In sharp contrast, CM from LBC cells significantly enhanced in vitro angiogenesis by ~6.5-fold above that of the responder cells or resting platelet controls (Fig. 3B).

Next, we tested whether platelets were capable of taking up pro-angiogenic factors released by instigating LBC tumor cells. We did so by co-culturing naïve, cancer-free mouse or human platelets with either responder or instigator cells in vitro. We interrogated the supernatants from these co-cultures for their ability to induce capillary tube formation, reasoning that if platelets absorbed proangiogenic factors from the medium, then supernatants from LBCs would have reduced angiogenic capacity following their exposure to platelets (Fig. 3C). Naïve platelets did not significantly alter the angiogenic ability of the responding tumor cell CM, which was negligible (Fig. 3B, D). Supernatants from LBC instigating cells, which were otherwise highly pro-angiogenic (Fig. 3B), exhibited ~15-fold and ~40-fold reductions in angiogenic ability when the LBC cells were cultured with naive mouse or human platelets, respectively (Fig. 3D). These results were consistent with previous reports (17) and established that both human and mouse platelets were capable of packaging pro- and anti-angiogenic factors secreted by LBC tumor cells.

In order to identify pro-angiogenic factors carried by platelets during LBC systemic instigation, we performed a human cytokine array on various platelet lysates. When compared to platelets from mice bearing responding tumors (no systemic instigation), a number of pro-angiogenic and pro-inflammatory human cytokines were significantly more concentrated in the platelets from mice bearing LBC instigating tumors, including GRO (p=0.012), IFNg (p=0.050), IL6 (p=0.044), PDGF-BB (p=0.033), and PlGF (p=0.044) (Fig. 3E). We validated these results by immunostaining platelets prepared from the various cohorts of mice (Fig. S4A-C). Notably, platelet-derived levels of vascular endothelial growth factor (VEGF) and thrombospondin (TSP), the most extensively studied pro-and anti-angiogenic cytokines, respectively, were not significantly different between cohorts (Fig. 3E and S4C).

In order to understand whether some of these cytokines were functioning at responding tumor sites, we examined the activation status of signal transducer and activator of transcription 3 (STAT3), which plays an important tumor-supportive role in both breast tumor cells and in the tumor microenvironment (23,24). STAT3 is a downstream effector of growth factor receptors for cytokines identified in our screen, including IL6 and PDGF (25). Using an antibody specific to the activated, phosphorylated form of both human and mouse STAT3 (p-STAT3), we noted that the levels of p-STAT3 were negligible in control responding tumors opposite Matrigel or TNBC (Fig. 3F). In marked contrast, p-STAT3 staining was abundant in responding tumors that grew contralaterally to LBC tumors, and was localized predominantly to the nucleus of stromal cells within these tumors (Fig. 3F). There was no significant difference in p-STAT3 levels when comparing BMCs from mice bearing the LBC instigating tumors to those of cancer-free controls (Fig. S4D), suggesting that the enhancement of STAT3 activity observed in the LBC-bearing mice did not occur in BMCs prior to their mobilization.

Taken together, these data established that LBC tumors loaded platelets with pro-inflammatory and pro-angiogenic factors and provided evidence that these factors were released at distant responding tumors sites. Despite equal concentrations of VEGF and TSP in platelets from both groups, platelets from LBC-bearing hosts had far greater pro-angiogenic activity, thus underscoring the importance of the complete repertoire of cytokine cargo carried by platelets under different pathological conditions.

BMC-LBC Mediate Enrichment of CD24+ Responding Tumor Cells

A paradigm of TNBC-mediated systemic instigation is that BMCs (specifically Sca1+/cKit- cells) are rendered pro-tumorigenic prior to mobilization from the marrow into the circulation; hence, when BMCs from hosts bearing TNBCs are admixed with responder cells prior to injection, the BMCs mimic the effects of the TNBC instigating tumors (8). We therefore tested whether the BMCs that were recruited into responding tumors (Fig. 1G, H) played an active role in the LBC systemic instigation process. To do so, we injected admixtures of responder cells and BMCs prepared from various mice and tested their tumor-promoting ability (Fig. 4A).

Figure 4.

BMCs from mice bearing LBC tumors enrich responding tumor cells for CD24 surface expression but lack instigating ability. A, In vivo test of BMC tumor promoting function. BMCs harvested from mice bearing indicated systemic environments were immediately mixed with responder cells and injected subcutaneously into secondary recipients. B, Tumor mass 12 wk following injection of responder cells admixed with indicated BMCs. Numbers of mice and incidence of tumor formation indicated below graph for collective data from 2 separate experiments. C and D, Responding tumors resulting from admixture with indicated BMCs stained for CD24 (green) (C), or CD24 (green) and pselectin (red) (D). Cell nuclei stained with DAPI (blue); scale bars = 100μm. E, Representative flow cytometry histograms of CD24 expression on GFP+ responder cells after 4 d in vitro co-culture with BMCs harvested from indicated mice. Gate represents CD24+ populations. Graph represents percent change in responding tumor cell CD24 surface expression under indicated conditions relative to co-culture with BMCs from cancer-free mice; n=10 BMC samples per group. F and G, VEGFR2-positive cells (red, F) and phospho-STAT3 (red) and LgT-positive tumor cells (green) (G) in responding tumors admixed with indicated BMCs; nuclei counterstained with DAPI (blue). H, Vessel density in indicated responding tumors; differences were not significant (n.s.). Also see Fig. S4.

BMCs prepared from mice bearing TNBC (BMC-TNBC) were sufficient for responding tumor growth (Fig. 4B). Responding tumors formed in 85% of these mice and tumors were ~2.8-fold larger than those that had formed on their own (40% incidence) or that had been admixed with BMCs from cancer-free hosts (68% incidence) (Fig. 4B). BMCs from mice bearing LBC tumors (BMC-LBC), however, did not significantly enhance incidence (68%) or mass of responding tumors above that of the cancer-free control BMCs (Fig. 4B). Nevertheless, consistent with the enrichment of CD24 on responder cells in the LBC environment (Fig. 2C, D), CD24 was likewise enriched on the surface of responding tumor cells that had been admixed with the BMC-LBCs (Fig. 4C), and p-selectin-positive platelet aggregates were localized to the CD24-rich areas of these tumors (Fig. 4D). The area covered by CD24+ cells in responding tumors admixed with BMCs from control mice was not as extensive as that of tumors admixed with BMC-LBC; consequently, fewer platelets were observed in the control tumors (Fig. 4C, D).

In order to understand whether BMCs from LBC tumor bearing mice directly mediated tumor cell surface enrichment of CD24, we cultured adherent GFP-positive (GFP+) responding tumor cells with BMCs prepared from various cohorts of mice and analyzed tumor cell expression of CD24 after 4 days by flow cytometry. Responding cells that had been cultured with BMCs harvested from TNBC-bearing mice (BMC-TNBC) displayed a ~30% decrease in CD24 expression (Fig. 4E), while exposure to BMCs from LBC-bearing mice (BMCLBC) resulted in a ~75% increase in the CD24-positive responding cell population relative to controls (Fig. 4E).

Hence, BMCs from mice bearing LBC tumors were necessary and sufficient to enrich CD24-positive responding tumor cells, which recruited platelets. Nevertheless, these events were not sufficient to enhance responding tumor malignancy. We therefore examined the resulting tumors for additional hallmarks of LBC-mediated tumor-promotion. While VEGFR2 cells were more abundant in the tumors that had been admixed with BMC-LBC, they did not appear to incorporate into tumor vasculature (Fig. 4F). The numbers of phosphorylated STAT3-positive cells, an indicator for the presence of pro-angiogenic platelets, were minimal and no different in the BMC-LBC admixed tumors than they were in the BMC-control admixed tumors (Fig. 4G). Consequently, there was no difference in tumor vessel density between the two cohorts (Fig. 4H) and in both cases, fewer vessels were apparent than in the tumor-promoting LBC systemic environment (Fig. 1E).

These findings suggested that without the stimulus provided by the LBC tumor, the platelets were not loaded with angiogenesis-promoting cargo and that instigating tumor-educated platelets were crucial for systemic promotion of responding tumor growth.

Instigating and Non-Instigating Primary Human Luminal Breast Cancers

In order to understand whether this type of systemic instigation processes might reflect real human tumor behavior, we tested 4 different primary human tumor specimens from patients with luminal breast cancer (hBRCA-LBC 1-4) for their ability to establish a pro-tumorigenic macroenvironment. To do so, we surgically implanted human tumor specimens or Matrigel control plugs subcutaneously into Nude mice contralaterally to responding human cells (Fig. 5A). These patient tumors were diagnosed as invasive ductal carcinoma, grade II/III, positive for ER and PR, with differing HER2 status, and each human tumor specimen retained its morphological characteristics and cytokeratin expression in the mice (Fig. S6A, B).

Figure 5.

Instigating, non-instigating, and responding human tumor specimens. A, Human luminal breast tumor (hBRCA-LBC) xenotransplantation model. Each of 4 surgical specimens (hBRCA-LBC 1 through 4) was implanted into 3 mice per cohort. B, Growth of responding tumors in environment established by hBRCALBC1 tumor specimens; n=3 mice. Inset: Ki67 (brown) of responding tumor formed in the hBRCA-LBC1 environment; nuclei counterstained with hematoxylin (blue). C, Responding tumors exposed to the non-instigating hBRCA-LBC2 or instigating hBRCA-LBC1 environments (top two rows); responding human LBC specimen (hBRCA-LBC 5) implanted into either instigating or control environments (bottom two rows). Tumors stained for phospho-STAT3 (red, column 1), VEGFR2 (red, column 2), CD24 (red, column 3), collagens (blue, column 4), and p-selectin (green, column 5); nuclei counterstained with DAPI (blue, columns 1, 2, 5), hematoxylin (blue, column 3), or Masson's Trichrome (red, column 4). Yellow arrows indicate blood vessels with exposed collagen; white arrows indicate blood vessels with intact endothelium. Scale bar = 100 μm for all images, except for p-selectin, where scale bar = 25 μm. D, Microvessel density of responding tumors in indicated hBRCA-LBC tumor environments. Tumors were examined under 40x magnification and 3 representative areas per tumor were analyzed; n=3 tumors (9 images) for Matrigel, n=3 tumors (9 images) for hBRCA-LBC1, n=1 tumor (3 images) for hBRCA-LBC2, no tumors were recovered opposite hBRCA-LBC3 or hBRCA-LBC4. E, Xenotransplantation model for responding human primay LBC (hBRCA-LBC 5); n = 5 mice per group. F, Growth kinetics of hBRCA-LBC5 implanted into either control Matrigel (black line) or LBC (red line) systemic environments. G, hBRCA-LBC 5 vessel density in tumors recovered from indicated environments. Tumors were examined under 40x magnification and 5 representative areas per tumor were analyzed. H, Human clear cell renal cell carcinoma (cRCC) xenotransplantation model. Each surgical specimen was implanted into 4 mice per cohort. I, Growth kinetics of responding cRCC specimens in the Matrigel control or LBC systemic environments as measured by tumor volume at indicated time points. J, Histopathologic features of human cRCC tissues recovered from mice bearing the LBC systemic environment (left) or in mice bearing Matrigel plugs (right). Top panels: hematoxylin and eosin (H&E) staining; Bottom panels: immunohistochemical labeling of CD34-positive endothelial cells; (X200 magnification). Inset: tumors cells within grafts grown in LBC tumor bearing mice express the human cRCC marker CAIX (X600 magnification). Also see Figs. S5, S6, S7.

One tumor, hBRCA-LBC1, established a pro-tumorigenic environment that supported growth of highly proliferative responding tumors in 100% (3/3) of the mice (Fig. 5B). This instigating tumor specimen exhibited identical growth kinetics whether it was implanted opposite Matrigel control or opposite the responding tumor cells (Fig. S6C). One (1/3) responding tumor was recovered opposite hBRCA-LBC2, none (0/3) were recovered opposite hBRCA-LBC3 or hBRCALBC4, and three (3/3) tissue plugs were recovered in the Matrigel control conditions.

All of the tumors that responded to hBRCA-LBC1 displayed hallmarks of LBC-mediated tumor promotion, including CD24 enrichment, p-selectin positive platelet aggregates, phospho-STAT3 positivity, and vessels marked by exposed collagen and incorporation of VEGFR2-positive cells when compared with the tumor recovered opposite non-instigating hBRCA-LBC2, in which enrichment of these hallmarks was not observed (Fig. 5C). Consequently, microvessel density in the responding tumors from the instigating hBRCA-LBC1 environment was significantly higher than those of the Matrigel (~3.4-fold) or non-instigating hBRCA-LBC2 (~7.2-fold) environments (Figs. 5D and S5D).

These results provided important evidence that primary tumor xenografts could be stratified based on their ability to establish a pro-tumorigenic systemic environment that promoted vascularization and growth of distant disseminated tumors.

Identification of Responding Tumors from Cancer Patients

Our results suggested that populations of cells that disseminate from a primary tumor in a patient with metastatic disease might respond to systemic signals to convert from a state of indolence to one of overt growth. To test this theory, we selected a tumor specimen from a patient with luminal breast cancer (hBRCA5, Fig. S6D) that remained indolent when implanted into tumor-free control mice (not shown). From this tumor, we prepared organoids (see Methods) and surgically implanted them beneath the skin of Nude mice bearing either instigating LBC tumors or control Matrigel plugs on the contralateral flanks (Fig. 5E).

All of the hBRCA5 organoids implanted into the LBC environment formed aggressively growing tumors that were significantly larger than the control organoids (Fig. 5F) and diaplayed all of the hallmarks of the LBC-mediated response. Specifically, the instigated hBRCA5 tumors were enriched for CD24, phospho-STAT3-positive cells, vessels with exposed collagen, VEGFR2-positive vessel cells, and p-selectin platelet aggregates compared with hBRCA5 organoids in the control environment (Fig. 5C, S6E). Moreover, the instigated tumors had a subtle yet significant increase (~38%) in vessel density than the counterpart control tissues (Fig. 5G).

Clear cell renal cell carcinoma (cRCC) is typically a highly vascularized cancer. In cRCC patients, levels of circulating VEGFR2+ progenitor cells correlate with outcome (26) and tumor cell enrichment of CD24 correlates with reduced progression free survival (27). We therefore tested the hypothesis that organoids prepared from a nephrectomy surgical specimen, taken from a patient with cRCC, would take advantage of the pro-angiogenic systemic macroenvironment established by instigating LBC tumors. cRCC organoids were surgically implanted beneath the skin of Nude mice bearing either instigating LBC or control macroenvironments (Fig. 5H). We selected one random organoid sample for histology to confirm that all animals received approximately equal portions of tumor tissue (Fig. S7A).

cRCC tissues recovered from mice bearing the LBC systemic environment were ~2-fold larger in volume and in mass than those that had been implanted opposite Matrigel control (Fig. 5I and S7B). Microscopic examination revealed the presence of tumor cell nodules in two of four grafts implanted in LBC tumor bearing mice but in none of the three grafts implanted in mice bearing Matrigel plugs (Figure 5J, top panels). Moreover, sparse cells with morphological features compatible with cRCC were detected in the other two LBC-instigated grafts that did not contain tumor nodules and in one of the grafts implanted opposite Matrigel control (not shown).

Immunohistochemical analysis demonstrated that the tumor cells composing the nodules within the LBC-instigated grafts expressed the cRCC marker Carbonic Anhydrase IX (CAIX) (Figure 5J, inset), and thus retained immunophenotypic features of the fresh tumor sample removed from the patient (Fig. S7A). Immunohistochemical labeling of mouse-derived CD34-positive cells revealed that one of the tumor nodules displayed high density of microvessels lined by murine endothelial cells compared with grafts implanted opposite Matrigel control (Fig. 5J, bottom panels).

Collectively these data indicated that human tumor breast cancer and clear cell renal carcinoma specimens that otherwise did not form successful grafts were able to take advantage of a pro-tumorigenic systemic environment to form vascularized, growing tumors.

Platelet Activity is Necessary for LBC-Mediated Systemic Instigation

In order to explore therapeutic potential and identify whether platelets were necessary for delivering the pro-tumorigenic instigating stimulus in the LBC macroenvironment, we treated mice with aspirin, which inhibits platelet activity (28). Mice were injected with LBC instigators that were permitted to grow for 4 weeks prior to initiation of weekly treatments of either aspirin or vehicle control; responders were then injected into these mice two days following the first aspirin treatment (Fig. 6A). While responding tumors formed in 90% of the mice treated with vehicle control, only 20% of the mice treated with aspirin developed responding tumors (Fig. 6B). Importantly, aspirin did not significantly affect instigating LBC tumor growth or circulating platelet counts in the two cohorts of mice (Fig. S8A, B).

Figure 6.

Aspirin treatment inhibits LBC-mediated systemic instigation. A, Experimental scheme to test effects of aspirin on LBC-mediated systemic instigation. All mice were injected with responders and LBC instigating tumors and treated with either 100 mg/kg aspirin or vehicle control; n=10 (5 mice per cohort for 2 independent experiments). B, Average mass of responding tumors recovered from indicated mice; incidence of tumor formation is indicated below graph. C, Responding tumors from indicated mice stained for VEGFR2 (red; top panels); pSTAT3 (red; bottom panels); nuclei counterstained with DAPI (blue). D, Numbers of VEGFR2+ cells in the bone marrow of experimental mice relative to those of cancer-free mice. Differences not statistically significant (n.s.). E, Flow cytometric analysis of CD24 expression on GFP+ responder cells co-cultured with indicated BMCs. Values represent percent increase in tumor cell CD24 levels using BMCs from indicated experimental mice relative to those using BMCs from cancer-free mice; n=3 per group; p values represent differences between indicated cohorts and control; values between the different conditions (aspirin v. vehicle) were not significant (n.s.). See also Fig. S8.

As expected, responding tumors growing contralaterally to instigating LBC from vehicle treated mice recruited VEGFR2-positive cells and were infiltrated with p-STAT3 positive stromal cells (Fig. 6C). In stark contrast, aspirin treatment completely inhibited the incorporation of VEGFR2-positive cells into responding tumor sites (Fig. 6C). These tissues were also negative for p-STAT3, suggesting that release of platelet-derived cytokines had not taken place at these tumor sites, as it had in the vehicle-treated controls (Fig. 6C).

Inhibition of responding tumor growth in response to aspirin was not due to a suppression of VEGFR2 cells in the marrow, as the numbers of VEGFR2-positive cells in the marrows of both vehicle-treated and aspirin-treated mice were ~2-fold higher than cancer-free mice and were not significantly different from one another (Fig. 6D). Moreover, aspirin treatment did not affect the ability of BMCs to enrich responding cell CD24 surface expression; tumor cells cultured with BMCs from either vehicle or aspirin treated mice had a ~90% increase in CD24 expression above those exposed to control BMCs from tumor-free mice, (Fig. 6E).

These results established that platelets mediated critical steps in the LBC-mediated systemic instigation cascade. Under instigating conditions, aspirin did not affect the instigating tumor or the activity of tumor-supportive VEGFR2+ cells in the marrow. Instead, platelet activity manifested most predominantly at the responding tumor site, where platelets were necessary for releasing proangiogenic cytokines and recruiting vessel-forming VEGFR2+ cells that facilitated the conversion from indolence to malignancy.

DISCUSSION

We describe a functional role for the systemic macroenvironment modulated by primary tumors that can ultimately determine growth and phenotype of secondary tumors (Fig. 7). In the presence of an instigating LBC tumor: i) circulating platelets are loaded with a repertoire of cytokines that significantly enhances their pro-angiogenic ability, and ii) the bone marrow is marked by an elevated number of VEGFR2+ cells. At the sites where otherwise indolent tumors reside, the proangiogenic platelets accumulate, most likely in response to exposed collagen and tumor cell CD24 presentation. Bone marrow cells play a two-part role: i) to provide VEGFR2+ cells that form the vasculature in response to pro-angiogenic factors, and ii) to mediate enrichment of tumor cell CD24, which can serve to recruit the pro-angiogenic platelets.

Figure 7.

Model of LBC-mediated systemic instigation. Instigating luminal breast cancers (LBC) establish a tumor-supportive host systemic macroenvironment by modulating circulating platelets and bone marrow cells. Circulating platelets are loaded with a repertoire of cytokines, derived at least in part from the LBC tumor, that render them pro-angiogenic. Platelets are recruited to sites where otherwise indolent tumors reside, ostensibly in response to exposed collagen IV as well as CD24 glycoprotein expression, where there is evidence that platelet-derived factors are released into the responding tumor microenvironent (e.g., activation of STAT3). Bone marrow cells, specifically VEGFR2+ cells, are present in elevated numbers in the marrow and are subsuquently mobilized to responding tumor sites where they contribute to the tumor vasculature. At the tumor site, BMCs enrich tumor cell surface expression of CD24, which can serve as a ligand for p-selectin expressed on platelets. This cascade of events results in the growth of highly vascularized responding tumors, which would have otherwise remained indolent. At present, the exact chronological sequence of these events is unclear. Aspirin treatment prevents responding tumor formation and disrupts recruitment of VEGFR2+ cells and activation of STAT3 in the responding tumors, without altering the numbers or function of VEGFR2+ cells in the marrow.

The significance of our results using instigating and responding primary human breast tumor specimens is supported by clinical observations that surgical resection of primary tumors improved the survival of women who presented with metastatic breast cancer at the time of diagnosis (29). Analysis of metastatic tumors from breast cancer patients demonstrated that CD24 expression is enhanced on tumor cells at metastatic sites relative to those in the primary tumor (30). CD24 has also been correlated with increased metastatic potential and reduced survival in both breast cancer (31,32) and cRCC (27) patients. Our study is the first to show that tumor cell enrichment of CD24 has important functional consequences and is directly driven by bone marrow derived cells – and not just any bone marrow cells, but only those from hosts bearing instigating luminal breast cancers.

cRCC is another example of a cancer for which surgical removal of the primary tumor (i.e., cytoreductive nephrectomy) improves patient outcome when performed prior to cytokine therapy (33,34). By demonstrating that human cRCC surgical specimens benefit from the macroenvironment established by instigating luminal breast cancers, we do not imply that the mechanisms of systemic instigation apply only to patients with concurrent breast and renal cell carcinoma. Rather, other cancer types, such as cRCC, might operate in a similar fashion to that of instigating luminal breast cancers to support the outgrowth of disseminated tumor cells. Indeed, systemic instigation processes might not only apply to the communication system between a primary tumor and its metastases, but between primary tumor foci (i.e, multifocal tumors), multiple primary tumors (i.e., contralateral breast cancer), or different metastatic colonies (7).

It has long been thought that in order for certain tumors to break dormancy, they must undergo an angiogenic switch (reviewed in (35)). In these earlier studies, the source of bioavailable pro-angiogenic factors at the metastatic sites was unclear. We found that platelets provide a rich source of these factors and change the perspective from one in which tumors intrinsically acquire angiogenic potential to one in which angiogenesis is the consequence of processes that are initiated at distant sites. A recent study in a large cohort of ovarian cancer patients established that paraneoplastic thrombocytosis is associated with reduced survival (36). Our study indicates that platelets might not be just an epiphenomenon of malignancy, but actual drivers of malignant progression.

Other studies have shown that interaction with platelets protects metastatic tumor cells from immune surveillance during their voyage in the circulation and helps tumor cells to attach to endothelium upon their arrest at metastatic sites (20). It was recently demonstrated that TGFβ in platelet releasates from cancer-free mice could enhance the metastatic potential of tumor cells (37). Our study expands upon these findings by demonstrating differences in the platelet cytokine repertoire between hosts bearing specific luminal breast tumors and cancer-free hosts, and establishing that platelets continue to promote cancer progression even after tumor cells have disseminated to distant sites. Indeed, platelets provide a major serum source of many pro-angiogenic proteins in the circulation of patients with breast (38) and colorectal cancer (39). Hence, our finding that both patelet cargo and function are quantitatively different in hosts bearing certain luminal breast cancers than they are in cancer-free individuals serves as a prerequisite for analyzing platelets from breast cancer patients.

Therapies targeting VEGF signaling have been less successful in the clinic than first anticipated (40), and approval to use Avastin, a monoclonal antibody against VEGF-A, in women with metastatic breast cancer was recently revoked by the FDA (41). Our study indicates that the repertoire of cytokines carried by platelets, including, but not limited to, VEGF, mediate pro-inflammatory and pro-angiogenic responses in the responding tumor microenvironment to promote tumor progression. Therefore, targeting these other factors, or the platelets that carry them, in the appropriate cohorts of patients should open new therapeutic windows.

Current guidelines suggest prophylactic aspirin use only with respect to cardiovascular protection; however, a role for aspirin in cancer prevention is also suggested from analysis of data from cardiovascular trials (42). Such analyses have established that aspirin use reduces the risk of cancer-related death, particularly from metastatic disease (43-46). Our data support the notion that a subset of breast cancer patients - those with instigating tumors - would benefit from aspirin use and emphasize the need for focused clinical trials because at present, there is not a consensus on whether aspirin use confers a survival benefit to breast cancer patients. A clear understanding of the molecular and cellular hallmarks that precisely define instigating luminal breast tumors and the pro-tumorigenic environment that they establish has the potential for more accurate selection of patients who would benefit from preemptive treatments, such as aspirin use.

METHODS

Cell Lines

Generation of HMLER-hygro-H-rasV12 (“HMLER-HR”), BPLER, and MCF7-Ras human mammary epithelial tumor cells has been previously described (11-13). Expression of cytokeratins and introduced oncogenes was validated for these studies; no additional authentication was performed by the authors.

Animals and Tumor Xenografts

Female Nude mice were purchased from Taconic (Hudson NY). All experiments were performed in accordance with regulations of the Children's Hospital Boston Institutional Animal Care and Use Committee (protocol 09-12-1566). Unless otherwise indicated, tumor cells were suspended in 20% Matrigel (BD Biosciences) and injected subcutaneously into nonirradiated mice. Tumors were measured on the flanks of live mice using calipers; volume was calculated as 0.5*length*(width²).

Bone Marrow Harvest and Transplantation

BMCs were harvested from donor mice by flushing femurs with sterile Hanks’ balanced salt solution (HBBS; GIBCO) with penicillin/streptomycin/fungisone. Cells were washed twice with sterile HBBS, dissociated with 18 g needle, and filtered through 70 μm nylon mesh. Bone marrow transplantation was performed as previously described (9) (see Supplemental Information).

Flow Cytometric Analysis

Fresh tissues were digested in 1 mg/ml collagenase A for 1–4 hr at 37° C with continuous rotation. Resulting cell suspensions were dispersed with an 18 g needle, washed twice with resuspension buffer (2% heat-inactivated fetal calf serum in sterile HBBS) and filtered through 70 μm nylon mesh. Cells were labeled for flow cytometry by incubation with appropriate antibodies for 30 min - 1 hr at 4° C with continuous rotation. Antibodies listed in Supplemental Information.

Immunohistochemistry

Dissected tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned onto ProbeOn Plus microscope slides (Fisher Scientific, Pittsburgh PA) for immunohistochemistry using Vectastain Elite ABC kits (Vector Laboratories, Burlingame CA) as previously described (9) or AlexaFluor fluorescenceconjugated antibodies (Invitrogen, Carlsbad CA). Antibodies listed in Supplemental Information.

Platelet preparations

Human and mouse platelets were isolated from whole blood by differential centrifugation as described (47) (detailed in Supplemental Information).

Angiogenesis Assay

Platelet releasates and cell conditioned media were tested for induction of human umbilical vein endothelial cells (HUVECs) capillary tube formation on Matrigel matrix in vitro using the In Vitro Angiogenesis Assay Kit (Millipore ECM625) according to manufacturer's instructions. Angiogenic ability was quantified by counting branchpoints, defined as the intersection of three or more capillary tubes. Branchpoints were counted each hour, from 4-7 hours and data represented as the average number of branchpoints per sample during the entire experimental time course.

Cytokine Array

Conditioned media or platelet lysates were tested on Human Angiogenesis Antibody Arrays (RayBiotech, Norcross GA) according to manufacturer's instructions.

Aspirin Treatment

450 mg Aspirin (Sigma, A2093-100G) was dissolved in 11 ml DMSO to make a 41 mg/ml concentrated stock solution. 30 min before injection, 1.6 ml of stock solution was diluted in 11.6 ml of PBS to make a 5 mg/ml injection solution.

Vehicle control was made with 1.6 ml DMSO diluted in 11.6 ml PBS. Mice were injected i.p. with aspirin at 100 mg/kg, once per week. A similar volume:weight ratio of vehicle was administered to control animals.

Human Breast Cancer and Renal Cell Carcinoma Tumor Specimens

Primary breast tumors were collected and processed shortly after resection in compliance with a protocol approved by the Brigham and Women's Hospital (IRB 93-085). Each tumor was analyzed for ER/PR/HER2 status. cRCC surgical specimens were obtained with patient consent from the Department of Pathology in compliance with a protocol approved by Brigham & Women's Hospital, Boston MA (DFCI IRB # 01-130). All specimens were used without patient identifiers. Tumors were cut to 3-4mm pieces, washed in RPMI, and frozen in RPMI + 10% DMSO. For xenograft studies, tumor specimens were quickly thawed at 37°C, washed 3 times in RPMI, and minced finely into <1mm organoids to ensure tissue homogeneity. Organoids were divided into equal portions, transferred to individual wells of a 96-well plate, covered with 50% Matrigel in RPMI media, and incubated for 10 min at 37°C to form cohesive plugs. One organoid plug was selected at random to confirm that samples contained viable tumor cells. Remaining organoid plugs were surgically implanted beneath the skin of Nude mice following sterile surgical procedure as previously described (9).

Statistical Analyses

Data are expressed as mean ± SEM. Data were analyzed by Student's t-test and were considered statistically significant if p < 0.05.

STATEMENT OF SIGNIFICANCE.

Currently, processes that mediate progression of otherwise indolent tumors are not well understood, making it difficult to accurately predict which cancer patients are likely to relapse. Our findings highlight the macroenvironment as an important component of disease progression that can be exploited to more accurately identify patients who would benefit from adjuvant therapy.

Abbreviations

LBC

Luminal breast cancer

TNBC

Triple-negative breast cancer

BMCs

Bone marrow cells

ER

Estrogen receptor

PR

Progesterone receptor

VEGFR2

Vascular endothelial growth factor receptor 2

PSTAT3

Phosphorylated form of the signal transducer and activator of transcription 3

BV

Blood vessel