Intratumoral heterogeneity impacts the response to anti-neu antibody therapy.

BACKGROUND: Along with de novo resistance, continued exposure to trastuzumab, an anti-human epidermal growth factor receptor 2 (HER2/neu) antibody, can lead to acquired resistance. In this study, we characterize a new anti-HER2/neu antibody resistant and metastatic mouse breast carcinoma cell line, TUBO-P2J. This cell line was developed during in vivo experiments using the antibody sensitive and non-metastatic tumor line TUBO. In addition, TUBO-P2J was used to establish an intratumoral HER2 heterogenous animal tumor model to evaluate the therapeutic effects of anti-HER2/neu antibody. METHODS: After establishing the cell line, TUBO-P2J was characterized regarding its susceptibility to anti-neu antibody and chemotherapeutics, as well as its metastatic potential in vitro and in vivo. In addition, expression profiles of metastasis related genes were also evaluated. A clinically relevant intratumoral HER2 heterogenous tumor model was established by inoculating mice with tumor cells consisting of TUBO and TUBO-P2J at a ratio of 1,000:1 or 10,000:1. Tumor growth and mouse survival were used to evaluate the therapeutic effects of anti-neu antibody. RESULTS: The TUBO-P2J cell line is a HER2/neu negative and highly metastatic variant of TUBO. This cell line was resistant to anti-neu antibody therapy, and when inoculated subcutaneously, metastasized to the lungs within 14 days. Compared to the parental TUBO cell line, TUBO-P2J displayed an epithelial-mesenchymal transition (EMT) related gene expression profile including: the loss of E-cadherin, and increased Vimentin, Snail, and Twist1 expression. In addition, TUBO-P2J exhibited increased invasion and migration activity, and was resistant to chemotherapy drugs. Finally, mixed tumor implantations experiments revealed that an increased percentage of TUBO-P2J rendered tumors less responsive to anti-neu antibody therapy. CONCLUSION: This study describes a novel model of intratumoral heterogenous metastatic breast cancer in immune competent mice that can be used to develop novel or combined immunotherapies to overcome antibody resistance.

Recipient NK cell inactivation and intestinal barrier loss are required for MHC-matched graft-versus-host disease.

Previous studies have shown a correlation between pretransplant conditioning intensity, intestinal barrier loss, and graft-versus-host disease (GVHD) severity. However, because irradiation and other forms of pretransplant conditioning have pleiotropic effects, the precise role of intestinal barrier loss in GVHD pathogenesis remains unclear. We developed GVHD models that allowed us to isolate the specific contributions of distinct pretransplant variables. Intestinal damage was required for the induction of minor mismatch [major histocompatibility complex (MHC)-matched] GVHD, but was not necessary for major mismatch GVHD, demonstrating fundamental pathogenic distinctions between these forms of disease. Moreover, recipient natural killer (NK) cells prevented minor mismatch GVHD by limiting expansion and target organ infiltration of alloreactive T cells via a perforin-dependent mechanism, revealing an immunoregulatory function of MHC-matched recipient NK cells in GVHD. Minor mismatch GVHD required MyD88-mediated Toll-like receptor 4 (TLR4) signaling on donor cells, and intestinal damage could be bypassed by parenteral lipopolysaccharide (LPS) administration, indicating a critical role for the influx of bacterial components triggered by intestinal barrier loss. In all, the data demonstrate that pretransplant conditioning plays a dual role in promoting minor mismatch GVHD by both depleting recipient NK cells and inducing intestinal barrier loss.

Anti-HER2/Neu passive-aggressive immunotherapy.

Preclinical studies have established that CD8+ T cells are necessary for efficient immunotherapeutic regimens targeting v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2, best known as HER2/Neu). Recently, we extended upon these findings by demonstrating that anti-HER2/Neu therapy also requires CD4+ T cells and CD40/CD40L signaling within the tumor microenvironment. Our results add to mounting evidence demonstrating that adaptive immunity is crucial to the efficacy of conventional and targeted anticancer chemotherapeutics.

Effective anti-neu-initiated antitumor responses require the complex role of CD4+ T cells.

PURPOSE: Targeting oncogenic receptors with antibodies has been thought to suppress tumor growth mainly by interrupting oncogenic signals. Recently, the essential role for adaptive immunity, and CD8(+) T cells in particular, has been established as a major factor for anti-HER2/neu-mediated tumor regression. However, the role of CD4(+) T cells is still being defined. The purpose of this study was to explore whether and to what extent CD4(+) T cells are involved in mediating the effects of anti-HER2/neu therapy. EXPERIMENTAL DESIGN: The role of CD4(+) T cells was examined using a transplant model of the rat HER2/neu-overexpressing cell line TUBO. Tumor-bearing mice were treated with anti-neu therapy in conjunction with CD4 depletion or CD40L blockade. The effects of CD4 depletion on the antitumor response were examined by tumor growth analysis and enzyme-linked immunospot (ELISPOT). RESULTS: In addition to CD8(+) T cells, CD4(+) T cells are also essential for anti-neu antibody-mediated tumor regression, but B cells are not required. The role for CD4(+) cells is necessary throughout anti-neu therapy and not limited to helping CD8(+) T cells. Expression of IFN-γ is necessary for anti-neu therapy and IFN-γ induces MHC-II expression in TUBO cells promoting direct recognition by CD4(+) T cells. Furthermore, intratumoral depletion of CD4(+) T cells or blockade of the activating cell-surface protein CD40L inhibits the antitumor response. CONCLUSIONS: This study reveals the essential role of CD4(+) T cell for anti-neu-mediated tumor regression.

Adaptive Immune Responses and HER2/neu Positive Breast Cancer.

Oncogenic signaling, such as HER2/neu signaling, has been shown to play major role for tumorigenesis in a subset of breast cancer patients. The use of anti-HER2/neu antibody has not only revealed the mechanisms for HER2/neu signaling but also shown a therapeutic advantage of its blockade. Indeed, the use of trastuzumab has greatly improved the treatment of HER2-positive breast cancer. Although this therapy has been used in the clinic for over twenty years, recent data is still uncovering new mechanisms by which this antibody exerts its anti-tumor activity. In addition to an improved understanding of the molecular mechanisms by which this therapy inhibits growth of tumor cells, the discovery that anti-HE2/neu therapy initiates and requires the adaptive immune system is one of these new mechanisms. The presence of anti-HER2/neu initiated adaptive immunity gives credence to efforts targeted at stimulating the immune system in treating HER2 positive breast cancer. This review focuses on the role of the inflammatory response in HER2 positive breast cancer with particular emphasis on trastuzumab therapy.

Cetuximab-mediated tumor regression depends on innate and adaptive immune responses.

Epidermal growth factor receptor (EGFR) over-signaling leads to more aggressive tumor growth. The antitumor effect of Cetuximab, an anti-EGFR antibody, depends on oncogenic-signal blockade leading to tumor cell apoptosis and antibody dependent cell-mediated cytotoxicity (ADCC). However, whether adaptive immunity plays a role in Cetuximab-mediated tumor inhibition is unclear, as current xenograft models lack adaptive immunity and human-EGFR-dependent mouse tumor cell lines are unavailable. Using a newly developed xenograft model with reconstituted immune cells, we demonstrate that the Cetuximab effect becomes more pronounced and reduces the EGFR(+) human tumor burden when adaptive immunity is present. To further study this in a mouse tumor model, we created a novel EGFR(+) mouse tumor cell line and demonstrated that Cetuximab-induced tumor regression depends on both innate and adaptive immunity components, including CD8(+) T cells, MyD88, and FcγR. To test whether strong innate signals inside tumor tissues amplifies the Cetuximab-mediated therapeutic effect, Cetuximab was conjugated to CpG. This conjugate is more potent than Cetuximab alone for complete tumor regression and resistance to tumor rechallenge. Furthermore, Cetuximab-CpG conjugates can activate tumor-reactive T cells for tumor regression by increasing dendritic cell (DC) cross-presentation. Therefore, this study establishes new models to evaluate immune responses induced by antibody-based treatment, defines molecular mechanisms, and provides new tumor-regression strategies.

Targeting and utilizing primary tumors as live vaccines: changing strategies.

Tumor metastases and relapse are the major causes of morbidity and mortality in cancer. Although surgery, chemotherapy and/or radiation therapy can typically control primary tumor growth, metastatic and relapsing tumors are often inaccessible or resistant to these treatments. An adaptive immune response can be generated during these conventional treatments of the primary tumor, and presumably both the primary tumor and secondary metastases share many of the same or similar antigenic characteristics recognized by the immune system. Thus, when established, this response should be able to control metastatic growth and tumor relapse. This review summarizes the mechanisms by which antitumor immune responses are generated, and recent findings supporting the hypothesis that many therapies targeting primary tumors can generate antitumor adaptive immune responses to prevent metastases and tumor relapse.

The therapeutic effect of anti-HER2/neu antibody depends on both innate and adaptive immunity.

Anti-HER2/neu antibody therapy is reported to mediate tumor regression by interrupting oncogenic signals and/or inducing FcR-mediated cytotoxicity. Here, we demonstrate that the mechanisms of tumor regression by this therapy also require the adaptive immune response. Activation of innate immunity and T cells, initiated by antibody treatment, was necessary. Intriguingly, the addition of chemotherapeutic drugs, although capable of enhancing the reduction of tumor burden, could abrogate antibody-initiated immunity leading to decreased resistance to rechallenge or earlier relapse. Increased influx of both innate and adaptive immune cells into the tumor microenvironment by a selected immunotherapy further enhanced subsequent antibody-induced immunity, leading to increased tumor eradication and resistance to rechallenge. This study proposes a model and strategy for anti-HER2/neu antibody-mediated tumor clearance.