Tumoral Lymphocytic Infiltration and Expression of the Chemokine CXCL10 in Breast Cancers from the Ontario Familial Breast Cancer Registry

Abstract

Purpose

Breast carcinomas, including basal and hereditary cases, often present with a prominent tumoral lymphocytic infiltrate (LI). Chemokines could play a role in attracting these cells and contribute to tumor progression. We explored tumoral expression of CXCL10 and determined the relationship between CXCL10 and LI in a cohort of breast cancers.

Experimental Design

Using tissue microarrays of 364 breast tumors we evaluated expression of CXCL10 and its receptor, CXCR3, in relation to histopathologic features, biomarkers and lymphocyte markers. Additionally, we overexpressed CXCL10 and CXCR3 in MCF7 breast cancer cells and monitored T-lymphocyte migration and invasion.

Results

Forty-five percent of tumors expressed CXCL10 and a significant association was found with CXCR3 and LI. Further characterization of the LI revealed an association with CXCL10 expression for peritumoral CD4+ and CD8+ lymphocytes. CD8+ intratumoral lymphocytes, FOXP3+ Tregs and T-BET+ Th1 cells were associated with BRCA1 and basal tumors. Conditioned media from MCF7 cells overexpressing both CXCL10 and CXCR3 increased T-lymphocyte migration and invasion.

Conclusions

Our findings suggest that CXCL10 may act in a paracrine manner, affecting the tumor microenvironment, and in an autocrine manner, acting on the tumor cells themselves and may play a role in tumor invasiveness and progression. The CXCL10-CXCR3 axis can serve as a potential target in BRCA1 and basal breast cancers which present with a prominent LI and a poor prognosis.

Introduction

Important roles for chemokines have been demonstrated in inflammation, infection, injury, allergy, as well as in cancer(1). Aside from their role in recruiting leukocytes to the site of inflammation, chemokines are also known to function as regulators of cell proliferation, differentiation, migration and invasion. In gene expression profiling we have previously identified the chemokine, CXCL10 (Interferon-gamma inducible protein 10), as being significantly overexpressed in basal tumors as compared to estrogen receptor (ER) positive tumors (Andrulis et al., in preparation). Secreted by activated T-lymphocytes, endothelial cells, fibroblasts, monocytes, and keratinocytes, it attracts other immune cells, such as activated T-lymphocytes, NK cells, and monocytes, which express its receptor, CXCR3.

Studies have shown a role for CXCL10 as well as its receptor in the progression of certain cancers(2). Both ligand and receptor expression have also been detected in a number of human breast cancer cell lines suggesting that the ligand acts on cells within the breast tissue microenvironment, and also acts on the tumor cells themselves in an autocrine manner to promote tumor growth(3). Conversely, CXCL10 has also been shown to inhibit tumor progression by its recruitment of mononuclear cells in hepatocellular carcinoma(4), and through antagonizing angiogenic factors and endothelial cells in renal cell carcinoma(5).

Carriers of germline mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 have an increased lifetime risk of developing breast cancer(6). BRCA1-associated cancers are poorly differentiated, are typically negative for ER, progesterone receptor (PR) and HER2(7). In addition, BRCA1-associated cancers show a specific morphologic phenotype: high mitotic count, a prominent lymphocytic infiltrate (LI) and pushing margins(8–9); features that are shared with basal-like breast cancers. BRCA2-associated breast cancers have also been shown to have a prominent LI(10). Despite the presence of a prominent host immune response, patients with hereditary breast cancers do not fare better. Furthermore, basal tumors, in patients unselected for family history, behave aggressively(11–14). We hypothesize that chemokines play a role in attracting these immune cells, and once present, these cells contribute to tumor progression.

Given the prominent LI found in a large proportion of basal and familial breast cancers, we investigated the expression of CXCL10 in a cohort of pathologically well characterized breast cancers from the Ontario site of the Breast Cancer Family Registry and sought to determine the relationship between CXCL10 expression and tumoral LI as well as molecular biomarkers and other morphologic and clinical parameters. The make-up of the LI was further characterized by immunohistochemical staining for the T lymphocyte markers CD4, CD8, FOXP3, T-BET and for the B lymphocyte marker CD20, and determining the association of these markers with CXCL10 expression, as well as morphologic and clinical parameters. Additionally, we evaluated the tumoral expression of the CXCL10 receptor, CXCR3, using immunohistochemistry, to investigate a possible autocrine role for the ligand. This autocrine role was further investigated in-vitro by co-expressing CXCL10 and CXCR3 in the MCF7 human breast cancer cell line and using conditioned media from these cells to study the migration and Matrigel invasion of T-lymphocytes towards this chemotactic source.

Materials and Methods

Study Population

Three hundred sixty four subjects were selected from the Ontario site of the Breast Cancer Family Registry(15) including 58 with known BRCA1 germline mutations and 64 patients with known BRCA2 germline mutations detected as previously described(16–18).

Tumor Morphology

As part of the Breast Cancer Family Registry, tumors from consenting patients undergo a centralized pathology review by an expert in breast pathology, using a standardized pathology reporting form(19). Included in this review are assessment of established pathologic prognostic factors including size, nodal status, tumor type, tumor grade and its individual components as well as specific tumor morphologic features. From this database information on morphologic features was abstracted.

Mutational Analysis of BRCA1 and BRCA2

Mutational analysis for BRCA1 and BRCA2 was performed using an RNA/DNA-based protein truncation test with complementary 5’ sequencing or complete gene sequencing by Myriad Genetics. Mutations were confirmed by DNA sequencing, and were classified as deleterious if they were protein-truncating, missense mutations (rare), or splice-site mutations as defined by the Breast Informatics Consortium (http://research.nhgri.nih.gov/bic/).

Tissue Microarray Construction and Immunohistochemical Staining

Tissue microarrays (TMA) (Beecher Instruments, Sun Prairie, WI) were constructed using duplicate 0.6mm cores of tumor. Four-micron TMA sections were cut and used for immunohistochemical staining using methods as listed in Supplementary Table S1. Staining for hormone receptors (ER, PR), HER2, basal (CK5, CK14) cytokeratins, EGFR, p53, Ki-67, CD4, CD8, CD20, FOXP3, T-BET, CXCL10 and CXCR3 was performed. Microwave antigen retrieval was carried out in a Micromed T/T Mega Microwave Processing Lab Station (ESBE Scientific, Markham, Ontario, Canada). Sections were developed with diaminobenzidine tetrahydrochloride and counterstained in Mayer’s hematoxylin.

Interpretation and Scoring of Immunohistochemistry

CD4, CD8 and CD20 positive lymphocytes were scored as present or absent and categorized as peritumoral or intratumoral. The latter were defined as those lymphocytes located within the epithelial component of the carcinoma. Absolute counts of T-BET+ lymphocytes and FOXP3+ lymphocytes were performed and these were categorized as intratumoral (when within the epithelial nests or within close proximity (the distance between positive lymphocyte and tumor nest is equal or less than the size of one tumor cell)) or peritumoral (at a distance from the epithelial nests). For FOXP3 and T-BET an absolute count of 10 positive lymphocytes (within or within close proximity of the epithelial cell nests) was used as the cutoff for positivity (Supplementary Figure S3).

The remaining immunohistochemical stained sections was scored using Allred’s scoring method(20), which adds the intensity of staining (absent: 0, weak: 1, moderate: 2, and strong: 3) to the percentage of cells stained (none: 0, <1%: 1, 1–10%: 2, 11–33%: 3, 34–66%: 4 and 67–100%: 5) to yield a ‘raw’ score of 0 or 2–8. Nuclear staining was scored for ER, PR, p53, and Ki-67 using previously validated cut-offs for ER and PR (>2 = positive)(21–22) and p53 (>3 = positive)(23). A cut-off of 4 was used for Ki-67. Moderate to strong complete membranous staining was assessed for HER2 and the validated cut-off of ≥ 5 was used to indicate positivity with this antibody(24). Membranous and/or cytoplasmic staining was scored for the remaining antibodies and a score of ≥ 4 was arbitrarily considered positive for CXCL10 while a score of ≥ 3 was considered positive for CXCR3, only expression in tumor cells was evaluated. The raw score data were reformatted using a TMA deconvoluter software program(25) into a format suitable for statistical analysis. Interpretable scores were obtained on 79 to 92% of tumors. Tumors from each group were assigned to molecular subgroups based on previous publications(26–28). Tumors that were positive for HER2 protein overexpression were assigned to the HER2 group. Tumors that were negative for HER2 but positive for ER or PR were assigned to the luminal group. Tumors that were negative for HER2, ER and PR, and positive for one or more of the following: CK5, CK14 or EGFR, were assigned to the basal subtype.

Cell Culture

The MCF7 cell line was grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS (both from Gibco), 10µg/ml bovine insulin (Sigma), and 100µg/ml penicillin-streptomycin (Gibco). This cell line passed authentication and is 100% match to ATCC’s HTB-22 (MCF7) STR profile. Media for transfected cells was supplemented with 550µg/ml G418 (MultiCell Technologies). T-lymphocytes were grown in Iscove’s Modified Dulbecco’s Medium supplemented with 10% human serum (Gemini), 100µg/ml penicillin-streptomycin, 10µg/ml gentamicine (Gibco), 5.5×10⁻⁵ M β-mercaptoethanol (Sigma), and 200 U/ml recombinant human IL-2 (Chiron Corp.).

Gene Transfection

pC-CXCL10 and pC-CXCR3 constructs were created by inserting the full-length human CXCL10 and CXCR3 cDNA, respectively, into the multiple cloning site of the pcDNA 3.1 TOPO vector (Invitrogen). CXCL10 cDNA was amplified from the RPMI-8226 cell line, while the CXCR3 sequence was commercially purchased (Open Biosystems Inc.). MCF7 cells were co-transfected with pC-CXCL10 and pC-CXCR3, or the empty vector using FuGene transfection reagent (Roche). Transfected cells were selected for and maintained with 550µg/ml G418.

T-Lymphocyte Isolation and Activation

Fresh blood was collected from consenting healthy donors, and peripheral blood mononuclear cells (PBMC) were isolated using CPT Vacutainer tubes according to the manufacturer’s instructions (BD). CD4+ T-lymphocytes were further isolated from the PBMCs through positive selection using magnetic bead sorting with a cell type specific antibody according to the manufacturer’s protocol (Miltenyi Biotec). The lymphocytes were activated over a 15-day period. The cells were cultured in complete media supplemented with 200 U/ml IL-2, and 1µg/ml phytohemagglutinin was added on the first day of plating only. Flow cytometry was used to verify CXCR3 upregulation in the activated T-lymphocytes.

ELISA

A CXCL10 specific ELISA development kit was used to confirm CXCL10 overexpression in conditioned media from the CXCL10-CXCR3 cells according to the manufacturer’s instructions (Peprotech). Conditioned media was generated by plating 1×10⁷ cells in complete media in 10-cm plates for 24h followed by washing in serum free medium (SFM) and a further incubation in SFM for 24h. Conditioned media was then collected, centrifuged at 1000×g for 10min, and frozen for later use in ELISA or transwell assays.

Flow Cytometry

To evaluate the expression of CXCR3 on CXCL10-CXCR3 cells and activated CD4+ T-lymphocytes the cells were stained with anti-CXCR3-PE or an IgG PE isotype-matched control (BD Biosciences). Additionally, the CD4+ T-lymphocytes were co-stained with anti-CD4-FITC and anti-CD45RO-APC to confirm the positive selection and activation status (BD Biosciences). Cells were washed and fixed with 2% paraformaldehyde. Data were acquired on the FACScalibur analytic flow cytometer (Becton Dickinson) and analysis was done using FlowJo software (Tree Star Inc.). Gating was done using forward scatter and side scatter, and both the percentage of CXCR3 positive cells and the mean fluorescence intensity (MFI) for CXCR3 were determined.

Transwell Migration and Invasion Assays

For T-lymphocyte transwell assays 1×10⁵ cells/well in 0.5% BSA serum free media were loaded in triplicate into the top chambers of 24-well inserts (5.0µm pore size, Costar). The bottom well contained conditioned media from either CXCL10-CXCR3 or empty vector control cells. Plates were incubated at 37°C for 2h, at which point the cells from the underside of the filter and from the lower chamber were collected. The CyQuant NF cell proliferation assay kit was used to quantify cell number (Invitrogen). Cell invasion was determined by coating the filters with Matrigel prior to cell placement (BD Biosciences). For CXCL10 neutralization experiments 120ng/ml of anti-CXCL10 antibody (R&D Systems) was added to the conditioned media for 1h at 37°C prior to T-lymphocyte addition.

Statistical Analysis

Tumor expression associations were tested using Chi-Square or Fisher's exact test. For in-vitro experiments significance was tested using the student’s t-test. As a further exploration, the overall survival (OS-died from breast cancer) differences in the four combination groups of CXCL10/CXCR3 were studied as well as those in FOXP3 positive versus negative and T-BET positive versus negative tumors. The follow up data was to the end of November 24, 2011. Excluding the patients lost to follow-up and those with deaths, the minimum follow-up time was 12 months after surgery and the median follow-up time was 148 months. Patient status on November 24, 2011 determined OS time and censoring status. Kaplan Meier (K-M) curves in combination with log rank tests were used to compare survival of the groups. All tests were two-sided. A test with a P-value <0.05 was considered statistically significant. All statistical analyses were performed using SAS 9.2 software (SAS Inc., Cary, NC, USA) with P-values unadjusted for multiple testing and K-M plots were done by R statistical software version 2.3.0 (http://www.r-project.org/).

Study Approval

Approval of the study protocol was obtained from the research ethics boards of Mount Sinai Hospital and the University Health Network, Toronto and written informed consent was received from all study participants.

Results

Expression of CXCL10 and Clinicopathologic Parameters

Forty-five percent of tumors were positive for CXCL10 expression (Figure 1 and Supplementary Figure S1). The histopathologic features of the tumors in this cohort are provided in Supplementary Table S2. The relationship between the expression of CXCL10 and histopathologic characteristics of the tumors was examined using TMAs. CXCL10 expression was found to be significantly associated with the presence of LI (80.1% vs. 65.4%, P=0.0053) and with higher tumor grade (36.4% vs. 26.4%, P=0.0215) (Table 1). In addition, expression of CXCL10 was negatively associated with margin circumscription (81.7% vs. 69.8%, P=0.0189). No significant associations were identified between tumor expression of CXCL10 and the genetic subgroups (data not shown) or with tumor size, lymphatic invasion, mitotic score or lymph node metastasis (Table 1).

Figure 1.

(A) Invasive breast cancer showing a prominent tumoral lymphocytic infiltrate. (B) Negative (i) and positive (ii) CXCL10 tumoral expression. (C) Negative (i) and positive (ii) CXCR3 tumoral expression.

Table 1.

Association between CXCL10 and Clinicopathologic parameters

		CXCL10
Tumor Characteristic		Positive		Negative
		n	%	n	%	P*-value
Grade
	High	73	55.3	90	55.2	0.0215
	Moderate	48	36.4	43	26.4
	Low	11	8.3	30	18.4
Size (mm)
	0–20	78	59.1	92	56.8	0.6911
	> 20	54	40.9	70	43.2
Lymphatic Invasion
	Positive	60	45.4	61	37.7	0.1764
	Negative	72	54.6	101	62.3
Mitotic score
	1	54	40.9	55	33.8	0.2048
	2 or 3	78	59.1	108	66.2
Number of LN_Positive
	0	67	55.4	82	54.0	0.8143
	> 0	54	44.6	70	46.0
Margin Circumscription
	Positive	24	18.3	49	30.2	0.0189
	Negative	107	81.7	113	69.8
Lymphocytic Infiltrate
	Absent	26	19.9	56	34.6	0.0053
	Present	105	80.1	106	65.4

^*P-values are from Chi-square or Fisher's exact test and are not adjusted for multiple testing.

Association of Lymphocyte Markers with CXCL10 and Clinicopathologic Parameters

The cellular make-up of the LI was assessed by determining cell surface expression of T lymphocyte (CD4+ and CD8+) and B lymphocyte (CD20+) markers in the intratumoral and peritumoral tissue areas (Supplementary Figure S2 for CD4 and CD8 images). The presence of peritumoral CD4+ as well as CD8+ lymphocytes was positively associated with CXCL10 expression (80.0% vs. 43.4%, P=0.0002 for peritumoral CD4+ lymphocytes; 49.6% vs. 0%, P=0.01 for peritumoral CD8+ lymphocytes) (Supplementary Table S3). Intratumoral CD8+ lymphocytes were positively associated with both the BRCA1 subgroup (20.0% vs. 7.3%, P=0.02) and the basal subtype (31.9% vs. 9.4%, P<0.0001), and negatively associated with ER expression (48.1% vs. 28.0%, P=0.002) (data not shown). Intratumoral CD20+ lymphocytes were positively associated with the basal subtype (55.9% vs. 17.3%, p<0.0001), and negatively associated with ER expression (73.5% vs. 34.5%, P<0.0001); however, an association with CXCL10 was not seen (data not shown). Altogether, these results suggest that T lymphocytes, but not B lymphocytes, are attracted by CXCL10 to the tumor site.

Association of CXCL10 Expression with Molecular Biomarkers

The expression of CXCL10 was found to be associated with two features that are also shared with the basal molecular subtype - p53 expression (40.0% vs. 26.7%, P=0.0191) and a high proliferation index, as determined by Ki-67 (58.5% vs. 35.5%, P=0.0001) (Table 2). CXCL10 expression was not related to expression of ER, PR, HER2, CK5, CK14 or EGFR (Table 2), nor to molecular (HER2, luminal and basal) subgroups, as previously defined (data not shown).

Table 2.

Association between CXCL10 and Molecular Biomarkers

		CXCL10
Biomarker		Positive		Negative
		n	%	n	%	P*-value
ER
	Positive	78	59.1	88	56.4	0.6464
	Negative	54	40.9	68	43.6
PR
	Positive	58	44.3	81	51.9	0.1966
	Negative	73	55.7	75	48.1
HER2
	Positive	16	12.0	12	7.8	0.2347
	Negative	117	88.0	141	92.2
p53
	Positive	52	40.0	39	26.7	0.0191
	Negative	78	60.0	107	73.3
CK5
	Positive	40	31.2	36	22.9	0.1141
	Negative	88	68.8	121	77.1
CK14
	Positive	19	15.2	16	10.2	0.2050
	Negative	106	84.8	141	89.8
EGFR
	Positive	18	13.9	19	13.1	0.8570
	Negative	112	86.1	126	86.9
Ki-67
	Positive	76	58.5	55	35.5	0.0001
	Negative	54	41.5	100	64.5
CXCR3
	Positive	36	29.8	16	11.3	0.0002
	Negative	85	70.2	126	88.7

^*P-values are from Chi-square or Fisher's exact test and are not adjusted for multiple testing.

CXCR3 Expression and Association with Clinicopathologic Parameters and Molecular Biomarkers

Twenty-one percent of tumors were positive for CXCR3 expression (Figure 1 and Supplementary Figure S1). CXCR3 expression was associated with low tumor grade (60.8% vs.40.0%, P=0.0065) and a low mitotic score (51.7% vs. 30.8%, P=0.0102) (data not shown). Further analysis stratified by CXCL10 status revealed that CXCR3 expression was associated with low tumor grade (43.8% vs.13.6%, P=0.0118) only in the CXCL10- group. Furthermore, CXCR3 expression was negatively associated with lymphatic invasion (75.0% vs. 54.9%, P=0.0048) (data not shown). No significant association was found between CXCR3 expression and tumor size, lymph node metastasis, margin circumscription, LI, molecular biomarkers, or the molecular or genetic subgroups (data not shown).

Co-expression of CXCL10 and CXCR3 in Breast Cancers

We hypothesized that CXCL10 tumor expression might be related to expression of CXCR3 and found this to be the case (29.8% vs. 11.3%, P=0.0002) as shown in Table 2. On further analysis of tumors that co-expressed both CXCL10 and CXCR3, CXCL10+/CXCR3+ was significantly associated with CK14 expression (29.4% vs. 9.4%, P=0.0027); other clinicopathologic parameters did not show significant associations. We also examined expression of CXCL9 and CXCL11, the other ligands for the CXCR3 receptor, by RT-PCR but did not detect significant differences in expression in basal versus ER+ tumors.

FOXP3+ Expression and Association with Clinicopathologic Parameters and Molecular Biomarkers

In an effort to determine if the immune response observed is pro or anti-tumor we examined the expression of FOXP3 as an immunohistochemical method to identify regulatory T cells (Tregs). We found that 32% (73 out of 226) of the tumors were positive for FOXP3 (Supplementary Figure S3). An association with CXCL10 expression was not seen (Supplementary Table S4). This suggested that at least in terms of Treg cells, CXCL10 is not specific in recruiting this component of the immune system. However, FOXP3 expression was found to be significantly associated with the basal molecular subtype and BRCA1 genetic group (P=0.0002 and P=0.0015, respectively) (Supplementary Table S5), as well as morphologic features characteristic of such tumors including high grade (87.5% vs. 46.7%, P<0.0001), p53 expression (48.6% vs. 29.2%, P=0.0054), ER negativity (56.3% vs. 35.6%, P=0.0036), PR negativity (67.1% vs. 44.7%, P=0.0019), CK5 positivity (38.4% vs. 20.7%, P=0.005), EGFR positivity (26.4% vs. 6.1%, P<0.0001), and a moderate LI (44.4% vs. 13.2%, P<0.0001) (Supplementary Tables S4 and S6). These results suggest that FOXP3+ cells could also be an important component of the lymphocytic infiltrates of basal and BRCA1 tumors separate from the immune cells that are specifically chemoattracted by CXCL10.

T-BET+ Th1 Lymphocyte Expression and Association with Clinicopathologic Parameters and Molecular Biomarkers

To further characterize the CD4 positive LI in the tumors we performed immunohistochemical staining and determined the expression of the Th1-associated transcription factor T-BET as CXCL10 is mainly a Th1-type chemokine. We found that 19% (40 out of 209) of the tumors were positive for tumoral infiltration by T-BET-positive lymphocytes (Supplementary Figure S3). T-BET expression was also found to be associated with the basal molecular subtype and BRCA1 genetic group (P=0.0002 and P=0.0329, respectively) (Supplementary Table S5), as well as morphological features characteristic of such tumors including high grade (84.6% vs. 54.2%, P=0.0021), p53 expression (56.4% vs. 30.4%, P=0.0024), ER negativity (64.1% vs. 39.3%, P=0.0050), PR negativity (74.4% vs. 48.2%, P=0.0032), CK5 positivity (47.5% vs. 23.8%, P=0.0029), EGFR positivity (30.0% vs. 8.6%, P=0.0003), and a moderate LI (48.7% vs. 19.9%, P<0.0001) (Supplementary Tables S4 and S6). Additionally, when considering only those tumors that had Th1 cells present (T-BET positive), 62% (23 out of 37 tumors) were CXCL10 positive (p=0.1390). Even though not statistically significant, this result supports the Th1-type chemokine status of CXCL10, and suggests that CXCL10 could be specifically attracting this particular CD4+ cell type to the tumor.

Expression of CXCL10, CXCR3, FOXP3 and T-BET and patient outcome

Finally, in an exploratory analysis, overall survival (OS) differences were examined between patients that fall into the following groups: tumors expressing (i) ligand and receptor (CXCL10+/CXCR3+), (ii) either ligand or receptor (CXCL10+/CXCR3−; CXCL10−/CXCR3+) or (iii) neither ligand nor receptor (CXCL10−/CXCR3−). As shown in Figure 2A, in the CXCL10+/CXCR3+ group (n=35) we observed 13 deaths (37%) as compared to 54 (24%) in the other three groups (n=222). Patients with CXCL10+/CXCR3+ tumors exhibited poorer OS compared to the other three groups, although the difference did not reach significance (P=0.1421). Additionally, we examined OS differences in FOXP3 positive versus negative tumors and T-BET positive versus negative tumors. While FOXP3 was not associated with a survival difference, T-BET, despite its positive correlation with an aggressive phenotype, was associated with an improved prognosis (Figure 2B). This finding requires further study as it may identify a population of patients with tumors showing a basal-like phenotype (both sporadic and BRCA1-related) who could have a favorable prognosis.

Figure 2.

(A) Kaplan-Meier survival curves according to expression of CXCL10 and CXCR3. (B) Kaplan-Meier survival curves according to expression of T-BET.

Activated CD4+ T-lymphocytes Show Increased Migration and Invasion towards Conditioned Media from CXCL10-CXCR3 Expressing Cells

Since CXCL10 is both secreted by and mainly chemoattracts activated CD4+ T-lymphocytes, it was of interest to determine if these cells would show increased migration and invasion to conditioned media from CXCL10-CXCR3 cells. Both CXCL10 and CXCR3 expression vectors were created and simultaneously transfected into the MCF7 human breast cancer cell line, which we previously determined does not express either of the genes endogenously. ELISA was used to verify CXCL10 overexpression, while flow cytometry was used to verify CXCR3 overexpression (Supplementary Figure S4). After 2h of incubation conditioned media from CXCL10-CXCR3 cells was able to significantly increase the migration (Figure 3A) as well as Matrigel invasion (Figure 3B) of the CD4+ T-lymphocytes as compared to their migration and invasion towards empty vector conditioned media.

Figure 3.

Activated CD4+ T-lymphocytes show increased migration and invasion towards CXCL10-CXCR3 conditioned media. (A) Transwell migration (no Matrigel) of T-lymphocytes towards conditioned media from CXCL10-CXCR3 and empty vector cells. Mean +/− SE (n=9 replicates), *p=0.02. (B) Transwell invasion (with Matrigel) of T-lymphocytes towards conditioned media from CXCL10-CXCR3 and empty vector cells. Mean +/− SE (n=9 replicates), *p=0.004. (C) Transwell migration of T-lymphocytes towards conditioned media from CXCL10-CXCR3 and empty vector cells incubated without or with anti-CXCL10 antibody for 1h prior to start of 2h cell migration. Mean +/− SE (n=6 replicates), *p<0.05.

To show that the presence of CXCL10 in the CXCL10-CXCR3 conditioned media was contributing to these results an anti-CXCL10 antibody was used to neutralize the activity of CXCL10 in the media prior to starting the transwell migration. There was no effect on the number of cells that migrated towards empty vector media. However, there was a 33% decrease in the number of cells that migrated to CXCL10-CXCR3 media (Figure 3C). Even after neutralization there were still significantly more cells migrating to the CXCL10-CXCR3 media compared to the empty vector media suggesting that neutralization was not complete or other factors present in the media were still mediating this effect, though to a lower extent, since neutralizing CXCL10 activity did decrease the number of cells migrating.

Discussion

Few studies have examined the expression of CXCL10 and/or its receptor, CXCR3, in breast tumors(29–30). This is the largest study to date to examine this chemokine axis and its association with various clinical and pathologic parameters in invasive breast carcinoma and, to our knowledge, the only one to examine it in familial breast cancer. We found that CXCL10 expression was significantly associated with LI, suggesting that tumoral expression serves to attract the various CXCR3-expressing immune cells. When we further characterized the cellular make-up of the LI by determining CD4 and CD8 (T-lymphocyte) and CD20 (B-lymphocyte) cell surface expression we found that it was only the presence of peritumoral CD4+ and CD8+ T lymphocytes that was significantly associated with CXCL10 positivity, and no association was found with the presence of B lymphocytes. Our characterization of the LI also supported the increased immune cell infiltration known to exist in the basal subtype and with BRCA1 carcinomas since the presence of intratumoral CD8+ lymphocytes, as well as FOXP3+ Tregs and T-BET+ Th1 cells, was associated with both these groups. Such an association has also been found in other cancers. For example, Clarke et al. have shown that the presence of intraepithelial CD8+ T cells significantly correlated with mutation or loss of expression of BRCA1 in ovarian carcinoma(31). Additionally, Mahmoud et al. found the total number of FOXP3+ tumor cells in invasive breast cancer to correlate with higher grade, ER negativity, and the basal subtype(32). While we did find Tregs in the tumors, they were not associated with CXCL10 expression. However, their association with both BRCA1 and basal tumors suggests that they could also be an important component of the lymphocytic infiltrate of these tumors separate from the immune cells that are specifically chemoattracted by CXCL10. Studies suggest that within the tumor microenvironment Tregs may serve to suppress anti-cancer cell immunity and to have a tumor-promoting role in the progression of established tumors(33–34). Thus, the presence of these cells in these tumors could serve a pro-tumor role.

Our in-vitro results showing that conditioned media from MCF7 cells overexpressing CXCL10 and CXCR3 significantly increased both the migration and invasion of activated CD4+ T-lymphocytes as compared to empty vector media further supports the possibility that CXCL10 is responsible for the presence of CXCR3 positive immune cells. Our results also show that the presence of CXCL10 itself in the conditioned media may contribute to this increased migration. These findings are consistent with those of other studies. For example, CXCL10 expression was only found in hepatocellular carcinomas in which there was marked lymphocyte infiltration(4) and CXCL10 expression in melanoma metastases was found to be associated with CD8+ T-lymphocyte recruitment(35). Additionally, tumor infiltrating CD8+ T lymphocytes significantly increased with stage progression in a study of stage I–III breast cancer patients, and Matkowski et al. found that patients with high expression of CD4 or CD8 had distinctly worse cancer specific overall survival(36–37). In ductal cancers however increased CD4 lymphocyte infiltration was linked to more aggressive histological features, such as higher grade and ER-negative status, and in univariate but not multivariate analysis was associated with significantly shorter survival(38). Therefore, it is possible that in our cohort it is the intratumoral CD8+ T lymphocytes in particular that are contributing more to breast cancer progression, and these are the cell type we have found to associate with the basal molecular subtype and BRCA1 genetic group.

Lymphocytic infiltration within the cancer milieu has been shown to be of mixed significance. Some reports have shown LI to play an important role in anti-cancer immunity(39–41) with others demonstrating a role for immune cells in tumor progression(42–43). Studies have shown a role for CXCL10 in the proliferation of certain cell types that express endogenous CXCR3, including vascular pericytes and smooth muscle cells(44–45). We found CXCL10 tumor cell expression to be associated with higher grade and a high proliferation index. In contrast, expression of its receptor, CXCR3, was found to be associated with favorable prognostic factors. The prognostic significance of tumoral CXCR3 expression has been previously examined with varying results. In breast cancer, Ma et al.(30) have found CXCR3 expression to be associated with a poorer OS in early stage disease. Our apparently contradictory result may be due to the CXCR3 positive tumors not having the same extent of LI since we did not find an association with LI in those tumors, and thus the inflammatory microenvironment that may contribute to prognosis is not established. Furthermore, when we examined the OS in an exploratory analysis, a possible survival difference was suggested between the CXCL10+/CXCR3+ group and the other three groups of ligand/receptor expression. We have found that rather than down-regulating expression of its receptor, tumoral CXCL10 expression was positively associated with tumoral CXCR3 expression and, interestingly, the group of patients with both CXCL10 and CXCR3 positive breast cancer showed a worse prognosis. Therefore, it is possible that not only is CXCL10 acting to shape the tumor microenvironment via a paracrine circuit, but also that it is acting in an autocrine manner on the tumor cells themselves, and the presence of both ligand and receptor could contribute to tumor progression. However, since our study was exploratory, and the OS result did not reach statistical significance, further studies to include a larger number of tumor samples and multivariate analysis to control for other factors are necessary to support this hypothesis.

FOXP3 expression has been reported to be associated with both poor, good and neutral outcomes(46). We did not find FOXP3 to be associated with a survival difference and the effect of FOXP3 on outcome remains unclear. Of interest, we found that the presence of T-BET+ lymphocytes was associated with a good prognosis. Recently, Ladoire et al. reported that the presence of T-BET+ lymphocytes in peritumoral lymphoid structures post neo-adjuvant therapy was associated with improved survival in trastuzumab-taxane treated patients with HER2 positive breast cancer(47). Our novel finding requires further study as it may identify a population of patients with tumors showing a basal-like phenotype (both sporadic and BRCA1-related) who could have a favorable prognosis.

Ligand binding to the CXCR3 receptor leads to activation of the PI3K and MAPK pathways(44, 48) and previous work has shown that activation of these pathways by CXCL10 binding results in changes in cell shape via actin polymerization, increased cell motility, chemotaxis, migration, and invasion(44, 48–50). It is therefore possible that the CXCL10-CXCR3 axis is acting in an autocrine fashion to increase the migratory capacity of these co-expressing cancer cells and plays a role in their invasiveness. Interestingly, we found CXCL10 protein expression to be negatively associated with margin circumscription, suggesting that the tumoral expression of CXCL10 and/or the presence of the associated host inflammatory response, may contribute to increased invasiveness of the leading tumor edge, which, in turn, may contribute to cancer progression.

We have found that tumor expression of CXCL10 is associated with lymphocytic infiltration; this LI was shown to be composed of both CD4+ and CD8+ T lymphocytes, including Tregs and Th1 cells. Furthermore, we found that CXCL10 was associated with increased expression of its receptor on tumor cells, and that conditioned media from CXCL10 and CXCR3 co-expressing cells increased the migration and Matrigel invasion of T-lymphocytes towards this chemotactic source. Our data suggest that CXCL10 may act in both a paracrine manner, affecting the tumor microenvironment, as well as in an autocrine manner, acting on the tumor cells themselves. Our novel observation that T-BET expression is associated with a better outcome requires further study as it may identify a population of patients with tumors showing a basal-like phenotype (both sporadic and BRCA1-related) who could have a favorable prognosis. The CXCL10-CXCR3 axis can serve as a potential drug target for BRCA1 and basal breast cancers which present with a prominent lymphocytic infiltrate and a poor prognosis and are currently in need of targeted therapy.

Statement of Translational Relevance.

CXCL10 expression was detected in primary breast cancers and found to be associated with tumoral lymphocytic infiltrate and expression of its receptor, CXCR3. Analysis of the infiltrate revealed association of CXCL10 with peritumoral CD4+ and CD8+ lymphocytes, intratumoral CD8+ lymphocytes, as well as FOXP3+ Tregs and T-BET+ Th1 cells. The latter were associated with the BRCA1 subgroup and basal subtype. Overexpression of this chemokine axis in MCF7 cells increased in-vitro T-lymphocyte migration and invasion. These results suggest that CXCL10 may act in both a paracrine manner, affecting the tumor microenvironment, as well as an autocrine manner, acting on the tumor cells themselves. The host immune response is believed to be important in outcome in many cancers. The CXCL10/CXCR3 axis may play a role and serve as a potential drug target for BRCA1 and basal breast cancers which present with a prominent lymphocytic infiltrate and a poor prognosis.

List of Abbreviations

ER

Estrogen receptor

PR

Progesterone receptor

LI

Lymphocytic infiltrate

TMA

Tissue microarray

EGFR

Epidermal growth factor receptor

HER2

Human epidermal growth factor receptor 2

CK5

Cytokeratin 5

CK14

Cytokeratin 14

OS

Overall survival