Elizabeth A. Repasky

Systemic endocrine instigation of indolent tumor growth requires osteopontin.

The effects of primary tumors on the host systemic environment and resulting contributions of the host to tumor growth are poorly understood. Here, we find that human breast carcinomas instigate the growth of otherwise-indolent tumor cells, micrometastases, and human tumor surgical specimens located at distant anatomical sites. This systemic instigation is accompanied by incorporation of bone-marrow cells (BMCs) into the stroma of the distant, once-indolent tumors. We find that BMCs of hosts bearing instigating tumors are functionally activated prior to their mobilization; hence, when coinjected with indolent cells, these activated BMCs mimic the systemic effects imparted by instigating tumors. Secretion of osteopontin by instigating tumors is necessary for BMC activation and the subsequent outgrowth of the distant otherwise-indolent tumors. These results reveal that outgrowth of indolent tumors can be governed on a systemic level by endocrine factors released by certain instigating tumors, and hold important experimental and therapeutic implications.

Characterization of Heat Shock Protein 110 and Glucose-Regulated Protein 170 as Cancer Vaccines and the Effect of Fever-Range Hyperthermia on Vaccine Activity

Several studies have confirmed that certain stress proteins can function as potent vaccines against a specific cancer when purified from the same tumor. Recent studies of two long-recognized but unstudied stress proteins, heat shock protein (hsp) 110 and glucose-regulated protein (grp) 170, have shown them to be efficient peptide chain-binding proteins. The present investigation examines the vaccine potential of hsp110 and grp170. First, it is shown that prior vaccination with hsp110 or grp170 purified from methylcholanthrene-induced fibrosarcoma caused complete regression of the tumor. In a second tumor model, hsp110 or grp170 purified from Colon 26 tumors led to a significant growth inhibition of this tumor. In addition, hsp110 or grp170 immunization significantly extended the life span of Colon 26 tumor-bearing mice when applied after tumor transplantation. A tumor-specific cytotoxic T lymphocyte response developed in the mice immunized with tumor-derived hsp110 or grp170. Furthermore, treatments of the mice with bone marrow-derived dendritic cells pulsed with these two proteins from tumor also elicited a strong antitumor response. Last, we showed that mild, fever-like hyperthermic conditions enhance the vaccine efficiency of hsp110 as well as heat shock cognate 70, but not grp170. These studies indicate that hsp110 and grp170 can be used in hsp-based cancer immunotherapy, that Ag-presenting dendritic cells can be used to mediate this therapeutic approach, and that fever-level hyperthermia can significantly enhance the vaccine efficiency of hsps.

Fever and the thermal regulation of immunity: the immune system feels the heat

Fever is a cardinal response to infection that has been conserved in warm-blooded and cold-blooded vertebrates for more than 600 million years of evolution. The fever response is executed by integrated physiological and neuronal circuitry and confers a survival benefit during infection. In this Review, we discuss our current understanding of how the inflammatory cues delivered by the thermal element of fever stimulate innate and adaptive immune responses. We further highlight the unexpected multiplicity of roles of the pyrogenic cytokine interleukin-6 (IL-6), both during fever induction and during the mobilization of lymphocytes to the lymphoid organs that are the staging ground for immune defence. We also discuss the emerging evidence suggesting that the adrenergic signalling pathways associated with thermogenesis shape immune cell function.

Tumor cell apoptosis, lymphocyte recruitment and tumor vascular changes are induced by low temperature, long duration (fever‐like) whole body hyperthermia

A single treatment of low‐temperature, long‐duration, whole‐body hyperthermia of either severe combined immunodeficient (SCID) mice bearing human breast tumor xenografts or Balb/c mice bearing syngeneic tumors for 6–8 hr can cause a temporary reduction of tumor volume and/or a growth delay. In both animal model systems, this inhibition is correlated with the appearance of large numbers of apoptotic tumor cells. Because this type of mild heat exposure, comparable to a common fever, is not itself directly cytotoxic, other explanations for the observed tumor cell death were considered. Our data support the hypothesis that this hyperthermia protocol stimulates some component(s) of the immune response, which results in increased antitumor activity. In support of this hypothesis, increased numbers of lymphocyte‐like cells, macrophages, and granulocytes are observed in the tumor vasculature and in the tumor stroma immediately following this mild hyperthermia exposure. In Balb/c mice, an infiltrate persists in the tumor for at least 2 weeks. Using the SCID mouse/human tumor system, we found that both host natural killer (NK) cells and injected human NK cells were increased at the site of tumor following hyperthermia treatment. Experiments using anti‐asialo‐GM1 antibodies indicate that the tumor cell apoptosis seen in the SCID mouse appears to be due largely to the activity of NK cells, although additional roles for other immunoeffector cells and cytokines appear likely in the immunologically complete Balb/c model. Another interrelated hypothesis is that immunoeffector cells may have greater access to the interior of the tumor because we have observed that this treatment causes an obvious expansion in the diameter of blood vessels within the tumor and an increase in nucleated blood cells within the vessels, which persists as long as 2 weeks after treatment. Further study of the mechanisms by which mild hyperthermia exerts antitumor activity could result in this treatment protocol being used as an effective, nontoxic adjuvant to immunotherapy and/or other cancer therapies. J. Cell. Physiol. 177:137–147, 1998.

Identification of genes differentially over-expressed in lung squamous cell carcinoma using combination of cDNA subtraction and microarray analysis.

In order to develop effective vaccine products against human cancer, we are interested in identifying genes over-expressed in tumor cells. Through a combination of cDNA library subtraction and microarray technology, we identified seventeen genes preferentially expressed in lung squamous cell carcinoma, including four novel genes. To date, expression profiles of these genes were confirmed by Northern and/or real-time analysis, and several genes were also found to be expressed in head and neck squamous tumors. Thus, these combined methods represent a high throughput approach for identifying tumor specific genes. Furthermore, the report of characterization on these genes will allow them to be exploited for their diagnostic, prognostic, and therapeutic potentials including immunotherapy and antibody based anticancer therapy.

Baseline tumor growth and immune control in laboratory mice are significantly influenced by subthermoneutral housing temperature.

Significance We show that the mandated, subthermoneutral laboratory housing temperature, which is known to cause chronic, metabolic cold stress, induces suppression of the antitumor immune response and promotes tumor growth and metastasis. When mice are housed at thermoneutrality, there are fewer immunosuppressive cells with significantly enhanced CD8+ T cell-dependent control of tumor growth. These findings underscore the fact that investigating mouse models under a single set of environmental temperature conditions may lead to a misunderstanding of the antitumor immune potential. These data also highlight the need for additional study to determine how systemic metabolic stress modulates the functions of immune effector cells, particularly in tumor-bearing mice, and whether cancer therapies, including immunotherapy, are impacted by housing temperature. We show here that fundamental aspects of antitumor immunity in mice are significantly influenced by ambient housing temperature. Standard housing temperature for laboratory mice in research facilities is mandated to be between 20–26 °C; however, these subthermoneutral temperatures cause mild chronic cold stress, activating thermogenesis to maintain normal body temperature. When stress is alleviated by housing at thermoneutral ambient temperature (30–31 °C), we observe a striking reduction in tumor formation, growth rate and metastasis. This improved control of tumor growth is dependent upon the adaptive immune system. We observe significantly increased numbers of antigen-specific CD8+ T lymphocytes and CD8+ T cells with an activated phenotype in the tumor microenvironment at thermoneutrality. At the same time there is a significant reduction in numbers of immunosuppressive MDSCs and regulatory T lymphocytes. Notably, in temperature preference studies, tumor-bearing mice select a higher ambient temperature than non-tumor-bearing mice, suggesting that tumor-bearing mice experience a greater degree of cold-stress. Overall, our data raise the hypothesis that suppression of antitumor immunity is an outcome of cold stress-induced thermogenesis. Therefore, the common approach of studying immunity against tumors in mice housed only at standard room temperature may be limiting our understanding of the full potential of the antitumor immune response.

HSP110-HER2/neu chaperone complex vaccine induces protective immunity against spontaneous mammary tumors in HER-2/neu transgenic mice.

Heat shock proteins (HSPs) are shown to be strong immunoadjuvants, eliciting both innate and adaptive immune responses against cancers. HSP110 is related in sequence to HSP70 and is ∼4-fold more efficient in binding to and stabilizing denatured protein substrates compared with HSP70. In the present study we evaluated the ability of a heat shock complex of HSP110 with the intracellular domain (ICD) of human HER-2/neu to elicit effective antitumor immune responses and to inhibit spontaneous mammary tumors in FVB-neu (FVBN202) transgenic mice. The HSP110-ICD complex was capable of breaking tolerance against the rat neu protein and inhibiting spontaneous mammary tumor development. This vaccine induced ICD-specific IFN-γ and IL-4 production. Depletion studies revealed that CD8+ T cells were involved in protection against challenge with mouse mammary tumors, whereas CD4+ T cells revealed partial protection. Increased IgG2a Ab titer in the sera of tumor-free animals after vaccination and elevated CD4+ CD25+ regulatory T cells in the PBL of tumor-bearing animals suggested that IFN-γ-producing Th1 cells may be responsible for partial protection of CD4+ T cells against the mammary tumor challenge, whereas CD4+CD25+ regulatory T cells (Th2 cells) may suppress the antitumor immune responses. Together, these results suggest that HSP110-ICD complex can elicit effective IFN-γ-producing T cells against spontaneous mammary tumors and that up-regulation of CD4+ CD25+ regulatory T cells may prevent complete eradication of the tumor following immunotherapy.

IL-6 trans-signaling licenses mouse and human tumor microvascular gateways for trafficking of cytotoxic T cells

Immune cells are key regulators of neoplastic progression, which is often mediated through their release of cytokines. Inflammatory cytokines such as IL-6 exert tumor-promoting activities by driving growth and survival of neoplastic cells. However, whether these cytokines also have a role in recruiting mediators of adaptive anticancer immunity has not been investigated. Here, we report that homeostatic trafficking of tumor-reactive CD8+ T cells across microvascular checkpoints is limited in tumors despite the presence of inflammatory cytokines. Intravital imaging in tumor-bearing mice revealed that systemic thermal therapy (core temperature elevated to 39.5°C ± 0.5°C for 6 hours) activated an IL-6 trans-signaling program in the tumor blood vessels that modified the vasculature such that it could support enhanced trafficking of CD8+ effector/memory T cells (Tems) into tumors. A concomitant decrease in tumor infiltration by Tregs during systemic thermal therapy resulted in substantial enhancement of Tem/Treg ratios. Mechanistically, IL-6 produced by nonhematopoietic stromal cells acted cooperatively with soluble IL-6 receptor-α and thermally induced gp130 to promote E/P-selectin- and ICAM-1-dependent extravasation of cytotoxic T cells in tumors. Parallel increases in vascular adhesion were induced by IL-6/soluble IL-6 receptor-α fusion protein in mouse tumors and patient tumor explants. Finally, a causal link was established between IL-6-dependent licensing of tumor vessels for Tem trafficking and apoptosis of tumor targets. These findings suggest that the unique IL-6-rich tumor microenvironment can be exploited to create a therapeutic window to boost T cell-mediated antitumor immunity and immunotherapy.

Discovery of differentially expressed genes in human breast cancer using subtracted cDNA libraries and cDNA microarrays

Identifying novel and known genes that are differentially expressed in breast cancer has important implications in understanding the biology of breast tumorigenesis and developing new diagnostic and therapeutic agents. In this study we have combined two powerful technologies, PCR-based cDNA subtraction and cDNA microarray, as a high throughput methodology designed to identify cDNA clones that are breast tumor- and tissue-specific and are overexpressed in breast tumors. Approximately 2000 cDNA clones generated from the subtracted breast tumor library were arrayed on the microarray chips. The arrayed target cDNAs were then hybridized with 30 pairs of fluorescent-labeled cDNA probes generated from breast tumors and normal tissues to determine the tissue distribution and tumor specificity. cDNA clones showing overexpression in breast tumors by microarray were further analysed by DNA sequencing, GenBank and EST database searches, and quantitative real time PCR. We identified several known genes, including mammaglobin, cytokeratin 19, fibronectin, and hair-specific type II keratin, which have previously been shown to be overexpressed in breast tumors and may play an important role in the malignance of breast. We also discovered B726P which appears to be an isoform of NY-BR-1, a breast tissue-specific gene. Two additional clones discovered, B709P and GABAA receptor π subunit, were not previously described for their overexpression profile in breast tumors. Thus, combining PCR-based cDNA subtraction and cDNA microarray allowed for an efficient way to identify and validate genes with elevated mRNA expression levels in breast cancer that may potentially be involved in breast cancer progression. These differentially expressed genes may be of potential utility as therapeutic and diagnostic targets for breast cancer.

Potent Anticarcinoma Activity of the Humanized Anti-CD70 Antibody h1F6 Conjugated to the Tubulin Inhibitor Auristatin via an Uncleavable Linker

Purpose: The antitubulin agent monomethyl auristatin F (MMAF) induces potent antitumor effects when conjugated via protease cleavable linkers to antibodies targeting internalizing, tumor-specific cell surface antigens. Humanized 1F6 (h1F6) is a humanized monoclonal antibody targeting CD70, a member of the tumor necrosis factor family that is expressed on hematologic malignancies and carcinomas. Here, we tested h1F6–maleimidocaproyl (mc) MMAF conjugates, consisting of an uncleavable mc linker, for their ability to interfere with the growth of CD70-positive carcinomas. Experimental Design: To evaluate the optimal drug per antibody ratio, we conjugated either four or eight MMAF molecules to the cysteines that comprise the interchain disulfides of h1F6 and determined antitumor activities in vitro and in xenografted mice. The tumor types tested included glioblastoma, patient-derived renal cell carcinoma (RCC) cell isolates, and standard RCC tumor cell lines. Results: All h1F6-mcMMAF conjugates potently interfered with the growth of all carcinomas in vitro and resulted in complete responses of RCC tumors implanted orthotopically or s.c. in mice. In vitro, h1F6-mcMMAF(8) was generally more potent than h1F6-mcMMAF(4). However, h1F6-mcMMAF(4) displayed equal or better efficacy than h1F6-mcMMAF(8) when administered to tumor-bearing mice. Conclusions: We showed that h1F6-mcMMAF conjugates inhibited the growth of human carcinomas and that increased drug loading, while improving potency in vitro, did not substantially affect the pharmacodynamic and pharmacokinetic properties in vivo. Based on these findings, h1F6-mcMMAF(4), designated SGN-75, has been identified as a potential antibody-drug conjugate for clinical development.