Isolation of immune cells from primary tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors. Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4(+) T helper cells via adoptive transfer, promotes CD8(+) T cells to maintain pro-inflammatory effector functions. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.     

Lymphocytes infiltrating human ovarian tumors. I. Role of Leu-19 (NKH1)-positive recombinant IL-2-activated cultures of lymphocytes infiltrating human ovarian tumors

Tumor-infiltrating lymphocytes (TIL) were obtained from human ovarian tumors, expanded in the presence of IL-2 in culture and studied for cytotoxicity against fresh autologous and allogeneic ovarian carcinoma (CA) targets. TIL from ovarian tumors grew well in long term cultures, achieving from 8- to 682-fold expansion. TIL cultured with IL-2 were cytotoxic against both autologous and allogeneic fresh ovarian CA targets, and no specificity for autologous tumor could be demonstrated in any of the cultures. In all fresh TIL preparations, CD3+ lymphocytes were the major cell type and contained a high proportion (up to 51%) of activated (IL-2R+) cells as determined by two-color flow cytometry. Sorting of bulk TIL cultures followed by cytotoxicity assays identified the Leu-19+ cells, both CD3+ and CD3-, as effectors of cytotoxicity against autologous and allogeneic tumor cell targets. Cold target inhibition assays showed that allogeneic targets (both ovarian CA and a sarcoma) competed effectively with autologous ovarian CA targets for Leu-19+ effectors in TIL cultures. mAb to Leu-19 or Leu-2a did not block lysis of autologous targets by sorted effectors. OKT3 antibody augmented lysis of autologous targets by CD3+Leu-19- effectors only. These results show that non-MHC-restricted Leu-19+ effectors in cultures of TIL with 1000 U/ml of rIL-2 mediate lysis of autologous and allogeneic tumor cells. The CD3+Leu-19- cells, the main population in these cultures, do not mediate tumor lysis. To determine the phenotype of antitumor effectors in IL-2 cultures of TIL, cell sorting followed by functional assays are necessary.     

Lectins as markers for cells infiltrating human renal glomeruli

This study describes a simple method for detecting mononuclear cells in human renal glomeruli using labeled lectins as probes. The lectins used in this study showed prominent binding to different cell types among the nonresident glomerular cells but not to normal glomerular elements. Monoclonal antibodies against monocytes/macrophages (OKM 1), T-cells (OKT 11), suppressior/cytotoxic T-cells (OKT 8), and anti-lysozyme antibodies were used in double-fluorescence studies with the lectins in an attempt to identify the lectin-positive cells. The results indicate that Bandeiraea simplicifolia I isolectin and Lotus tetragonolobus agglutinin in particular are useful for screening of kidney biopsies for cells of their monocyte/macrophage series and T-cells invading human renal glomeruli.     

Gene expression programs of human smooth muscle cells: tissue-specific differentiation and prognostic significance in breast cancers

Smooth muscle is present in a wide variety of anatomical locations, such as blood vessels, various visceral organs, and hair follicles. Contraction of smooth muscle is central to functions as diverse as peristalsis, urination, respiration, and the maintenance of vascular tone. Despite the varied physiological roles of smooth muscle cells (SMCs), we possess only a limited knowledge of the heterogeneity underlying their functional and anatomic specializations. As a step toward understanding the intrinsic differences between SMCs from different anatomical locations, we used DNA microarrays to profile global gene expression patterns in 36 SMC samples from various tissues after propagation under defined conditions in cell culture. Significant variations were found between the cells isolated from blood vessels, bronchi, and visceral organs. Furthermore, pervasive differences were noted within the visceral organ subgroups that appear to reflect the distinct molecular pathways essential for organogenesis as well as those involved in organ-specific contractile and physiological properties. Finally, we sought to understand how this diversity may contribute to SMC-involving pathology. We found that a gene expression signature of the responses of vascular SMCs to serum exposure is associated with a significantly poorer prognosis in human cancers, potentially linking vascular injury response to tumor progression.     

Cytomegalovirus infection induces a stem cell phenotype in human primary glioblastoma cells: prognostic significance and biological impact

Glioblastoma (GBM) is associated with poor prognosis despite aggressive surgical resection, chemotherapy, and radiation therapy. Unfortunately, this standard therapy does not target glioma cancer stem cells (GCSCs), a subpopulation of GBM cells that can give rise to recurrent tumors. GBMs express human cytomegalovirus (HCMV) proteins, and previously we found that the level of expression of HCMV immediate-early (IE) protein in GBMs is a prognostic factor for poor patient survival. In this study, we investigated the relation between HCMV infection of GBM cells and the presence of GCSCs. Primary GBMs were characterized by their expression of HCMV-IE and GCSCs marker CD133 and by patient survival. The extent to which HCMV infection of primary GBM cells induced a GCSC phenotype was evaluated in vitro. In primary GBMs, a large fraction of CD133-positive cells expressed HCMV-IE, and higher co-expression of these two proteins predicted poor patient survival. Infection of GBM cells with HCMV led to upregulation of CD133 and other GSCS markers (Notch1, Sox2, Oct4, Nestin). HCMV infection also promoted the growth of GBM cells as neurospheres, a behavior typically displayed by GCSCs, and this phenotype was prevented by either chemical inhibition of the Notch1 pathway or by treatment with the anti-viral drug ganciclovir. GBM cells that maintained expression of HCMV-IE failed to differentiate into neuronal or astrocytic phenotypes. Our findings imply that HCMV infection induces phenotypic plasticity of GBM cells to promote GCSC features and may thereby increase the aggressiveness of this tumor.GBM is the most prevalent and the most aggressive primary malignancy of the central nervous system in adults. It is a highly vascularized and infiltrating tumor, rarely cured and prone to recurrence. The median duration of survival after diagnosis is less than 15 months, despite aggressive therapy consisting of surgical resection and concomitant radiotherapy and chemotherapy.¹ Surgical resection of GBMs is typically incomplete, as they are located in the brain and are highly infiltrative. Postoperative radiotherapy and chemotherapy fail to eradicate all remaining GBM cells. Thus, a breakthrough in identifying a new treatment option leading to a cure of this disease is still lacking.GBMs contain a subpopulation of highly tumorigenic cells with unlimited capacity for self-renewal that are commonly resistant to standard therapy. Phenotypically and functionally, these cells resemble neural stem cells and, when implanted in immunodeficient mice, can generate new tumors. As a result, they are referred to as glioma cancer initiating cells or glioma cancer stem cells (GCSCs) (reviewed in Lima et al.²). Because of their apparent pivotal role in gliomagenesis and tumor recurrence after therapy, GCSCs are a major focus of research whose ultimate goal is to identify more effective therapies for GBM patients.GCSCs were first identified by their surface expression of CD133, based on the findings that these cells grow as neurospheres under nonadherent conditions and that tumors form in vivo after implantation of only 100 CD133-positive GBM cells but not after implantation of 10⁵ CD133-negative GBM cells.³ The importance of CD133 as a marker of tumor aggressiveness was corroborated by the correlation between CD133 expression in brain tumors and a poor clinical prognosis.^{4, 5, 6} However, later studies revealed that CD133-negative cells can give rise to CD133-positive cells^{7, 8, 9} and that both CD133-positive and CD133-negative GBM cells can initiate the development of highly aggressive tumors.¹⁰ Moreover, diverse GCSC types – all capable of self-renewal and tumor initiation – coexist within the same GBM.¹⁰ These cells often express markers associated with stem cells such as Sox2, Notch, and Oct-4.^{11, 12, 13} This intratumoral heterogeneity and the resulting aggressiveness of GBMs are influenced by the location of the tumor within the brain and by tumor-associated microenvironmental factors (reviewed in Stopschinski et al.¹⁴). While the general validity of CD133 as a major GCSC marker is still debated and its exact function in gliomagenesis remains poorly understood, other GCSC markers have been identified, including Notch1, Oct4, Sox2, and Nestin.^{4, 14, 15} The high levels of expression of these markers appear to functionally induce or maintain features that are characteristic of GCSCs.We have focused on developing and testing novel treatments for GBM based on two observations: that 99% of GBMs contain human cytomegalovirus (HCMV) proteins^{16, 17} and nucleotide sequences, and that the degree of HCMV protein expression in GBMs is a prognostic factor for patient survival.¹⁸HCMV is a herpesvirus that infects 70–100% of the world''s population. After an active primary infection, usually asymptomatic or subclinical in immunocompetent individuals, the virus establishes latency in the bone marrow and peripheral blood. Latent infections can be reactivated by inflammation. In immunocompromised individuals, primary HCMV infection and reactivation are significantly associated with morbidity and mortality.^{19, 20}In a clinical trial, we found that the antiviral drug valganciclovir as an add-on to standard therapy led to high survival rates among GBM patients. In a retrospective analysis of patients continuously receiving such therapy for more than 6 months, the 2-year survival rate was 90% and median overall survival was 56.4 months, as compared with 18% and 13.5 months, respectively, in contemporary controls.¹⁷ These results suggest that HCMV has an oncogenic or an oncomodulatory role in GBMs, and highlight the possibility that valganciclovir may eliminate or modulate the behavior of GCSCs that may not be targeted with conventional therapies.In light of these findings, we hypothesized that HCMV infection of GBM cells and the maintenance of a GCSC phenotype could be interrelated events. To test this hypothesis, here we investigated potential co-expression of a GCSC marker with HCMV immediate-early protein in a series of human clinical GBM specimens, and experimentally assessed the ability of HCMV infection to induce a GCSC phenotype in primary human GBM cells.     

WSX1 expression in tumors induces immune tolerance via suppression of effector immune cells

Crosstalk between tumor cells and the cognate microenvironment plays a crucial role in tumor initiation and progression. However, only a few genes are known to affect such a crosstalk. This study reveals that WSX1 plays such a role when highly expressed in tumor cells. The expression of WSX1 in Lewis Lung Carcinoma (LLC) and the melanoma cell line AGS induces the death of T cells and inhibits the production of the effector cytokine IFNγ from NK and T cells, resulting in the promotion of tumor growth. These pro-tumorigenic properties of WSX1 are independent of IL27. This key observation reveals a new pathway of tumor-host interaction, which will ultimately lead to better strategies in immune therapy to reverse tumor tolerance.     

Expression of serum miR-221 in human hepatocellular carcinoma and its prognostic significance

Objective

To investigate whether the serum miR-221 expression correlates with clinicopathologic features and the prognosis of hepatocellular carcinoma (HCC) patients.

Methods

Four miRNAs (miR-221, miR-222, miR-21 and miR-224) related to HCC were selected in the present study. Serum miRNA expression was investigated in 46 HCC patients and 20 healthy normal controls by using real-time PCR technique, and then correlations between miR-221 expression and the clinicopathological features and prognosis of HCC patients were evaluated.

Results

The four miRNAs were found to be differentially overexpressed in HCC serum samples, and high level of miR-221 expression was correlated with tumor size (P < 0.001), cirrhosis (P = 0.003) and tumor stage (P = 0.016). In addition, Kaplan–Meier survival analysis showed that the overall survival rate of the high miR-221 expression group (27.6%) was significantly lower than that of the low miR-221 expression group (62.3%, P < 0.05).

Conclusions

Serum miR-221, upregulated in HCC, can provide predictive significance for prognosis of HCC patients.     

Superoxide dismutases in malignant cells and human tumors

Reactive oxygen metabolites have multifactorial effects on the regulation of cell growth and the capacity of malignant cells to invade. Overexpression of the superoxide dismutases (SODs) in vitro increases cell differentiation, decreases cell growth and proliferation, and can reverse a malignant phenotype to a nonmalignant one. The situation in vivo is more complex due to multiple interactions of tumor cells with their environment. Numerous in vivo studies show that the superoxide dismutases can be highly expressed in aggressive human solid tumors. Furthermore, high SOD has occasionally been associated with a poor prognosis and with resistance to cytotoxic drugs and radiation. Most of the apparent conflicts between the above in vitro and in vivo observations can be reconciled by considering the net redox status of tumor cells in different environments. Administering high concentrations of SOD to cells in vitro is usually associated with a non- or less malignant phenotype, whereas secondary induction of SOD in tumors in vivo can be associated with an aggressive malignant transformation probably due to the altered (oxidative) redox state in the malignant cells. This concept suggests that for many types of tumors antioxidants could be used to diminish the invasive capability of malignant cells.     

Interaction of human multipotential mesenchymal stromal and immune cells

Multipotential mesenchymal stromal cells (MMSCs) are the subject of increasing scientific interest due to their key role in physiological renewal and repair. Allogeneic MMSCs interaction with other components of tissue environment, in particular with immune cells, represent one of the most intriguing question of modern cell physiology. MMSCs possess pronounced immunomodulatory capabilities based on their "immmunopriveledge" properties and the ability to suppress immune response. This review is highlighted the current state of art in the field of MMSCs immunomodulatory effects realization and mechanisms. MMSCs and immune cells interaction represents complex multidirectional process governed by both direct cell-to-cell interactions and soluble factors (interferon-gamma, tumor necrosis factor, prostaglandin E2, hepatocyte growth factor, interleukins ets.). The importance of physical environmental factors, primarily oxygen tension, on peculiarities of MMSCs and immune cells interaction is discussed.     

Expression of microtubule-associated protein TPX2 in human gastric carcinoma and its prognostic significance

Background

The molecular mechanisms underlying the development and progression of gastric carcinoma remain poorly understood. The main objective of this study was to investigate the expression level of targeting protein for Xenopus kinesin-like protein 2 (TPX2) and its clinical significance in human gastric carcinoma.

Methods

Real-time quantitative polymerase chain reaction (RT-PCR) and western blotting were used to determine the mRNA and protein levels of TPX2 in 20 paired gastric carcinoma tissues and the adjacent normal tissues, and the expression of TPX2 protein in 106 specimens of a gastric carcinoma tissue microarray was determined by immunohistochemistry. The associations of TPX2 expression with the clinicopathological features were analyzed, and the prognosis of gastric carcinoma patients was evaluated.

Results

The results showed that the expression of TPX2 mRNA was significantly higher in gastric carcinoma than in the adjacent normal tissues in 20 paired samples. Western blotting analysis revealed that TPX2 protein was differentially increased in 17 of 20 specimens from primary human gastric carcinoma tissues compared with those from adjacent non-tumor tissues. Immunohistochemical staining showed that TPX2 over-expression was significantly associated with advanced age (P = 0.001) and tumor T stage (P = 0.003). In addition, TPX2 was an independent prognostic factor for overall survival (OS) in the multivariate analysis [hazard ratio (HR) 0.001; 95 % confidence interval (CI) 2.626–7.198; P = 0.001].

Conclusions

TPX2 is up-regulated in gastric carcinoma and is associated with old age and tumor T stage. TPX2 may serve as a good prognostic indicator in patients with gastric carcinoma.

     

Isolation and characterization of human papillomavirus type 6-specific T cells infiltrating genital warts.

The potential role of T cells in the control of human papillomavirus type 6 (HPV-6) infections is an appealing premise, but their actual role has been sparsely investigated. Since HPV-6 infections are confined to the epithelium, such an investigation should focus on the T cells present at the site of infection (i.e., the warts). Therefore, we isolated wart-infiltrating lymphocytes (WIL) from patients with clinically diagnosed anogenital warts. These WIL were characterized by their phenotype and their specificity for E7 and L1 proteins of HPV-6. The phenotype of WIL varied drastically from patient to patient, as determined by their expression of CD4, CD8, T-cell receptor alpha/beta chain (TCR alpha beta), and TCR gamma delta. Despite this heterogeneity in phenotype, HPV-6 E7 and/or L1-specific WIL, as determined by lymphoproliferation, could be isolated from more than 75% of the patients studied. Among all L1 peptides recognized by WIL, peptides 311-330 and 411-430 were the most consistently detected, with seven of nine patients for whom L1 peptide reactivity was observed responding to at least one of them. Moreover, the HPV-6 epitopic peptides recognized by WIL differed to some extent from those recognized by peripheral T cells.     

Tumor infiltrating lymphocytes of spontaneous versus transplanted mouse mammary tumors

Summary Tumor infiltrating lymphocytes (TIL) were isolated by centrifugal elutriation from C4 mouse mammary tumors and characterized with regard to phenotype and natural killer (NK) activity. Tumors that had arisen spontaneously in prenoplastic hyperplastic alevolar nodules and tumors that had been passaged one to two times in either naive or presensitized mice were studied. Mice were sensitized by limited s.c. tumor growth and subsequent surgical removal of the tumor. The total numbers of T or B cells in the infiltrates were similar in spontaneous tumors and in passaged tumors from either naive or sensitized mice. The ratio of L3T4-positive to lyt-2-positive cells was reduced, however, from 1.10±0.2 in spontaneous tumors to 0.53±0.28 or 0.48±0.04 in passaged tumors from untreated or sensitized mice. The site of tumor implantation, whether intramammary fat pad or s.c., did not affect the profiles of the infiltrates. The TIL from both spontaneous and passaged tumors demonstrated enhanced NK activity relative to peripheral lymphoid cells. The TIL of passaged tumors sensitized mice, however, had lower NK activity than those from naive mice.     

Functional analysis of mononuclear cells infiltrating into tumors. IV. Purification and functional characterization of cytotoxic cell-generating factor

Rat cytotoxic cell-generating factor (CGF) was purified from cell-free supernatants of a T cell hybridoma (6B2-B8) that constitutively produces CGF. CGF activity was assessed by its ability to generate cytotoxic cells against 51Cr-labeled T-9 cells from spleen cells of T-9-immunized rats. The purification scheme consisted of ammonium sulfate precipitation, AcA 54 gel permeation, Mono Q anion exchange chromatography, Superose 12HR 10/30 gel permeation, SDS-PAGE with subsequent electroelution, and ProRPC HR5/10 reverse phase column chromatography. Overall, CGF was purified approximately 13,000-fold, with a maximum 2.5% recovery of activity, and the sp. act. of the purified CGF was approximately 19,000 U/mg. The purified CGF is distinct from the other lymphokines such as IL-1, IL-2, IL-3, IL-4, T cell-replacing factor/IL-5, IL-6, and IFN-gamma. It is capable of promoting the generation of cytotoxic T cells from R1-10B5 (+) spleen cells of T-9-immunized rats and also stimulates a W3/25 (+) T cell hybridoma to express the IL-2R. The CGF has an apparent m.w. of 28,000 under non-reducing and 14,000 and 16,000 under reducing conditions. 125I-labeled CGF binds to normal thymocytes as well as splenic T cells. The highest level of binding of CGF was detected on splenic T cells derived from T-9-immunized rats that were previously shown to contain CTL precursors. The binding analysis with 125I-labeled CGF demonstrated that CGF binds to a specific cell surface molecule with an approximate m.w. of 60,000 to 70,000.     

Early T cell signalling is reversibly altered in PD-1+ T lymphocytes infiltrating human tumors

To improve cancer immunotherapy, a better understanding of the weak efficiency of tumor-infiltrating T lymphocytes (TIL) is necessary. We have analyzed the functional state of human TIL immediately after resection of three types of tumors (NSCLC, melanoma and RCC). Several signalling pathways (calcium, phosphorylation of ERK and Akt) and cytokine secretion are affected to different extents in TIL, and show a partial spontaneous recovery within a few hours in culture. The global result is an anergy that is quite distinct from clonal anergy induced in vitro, and closer to adaptive tolerance in mice. PD-1 (programmed death -1) is systematically expressed by TIL and may contribute to their anergy by its mere expression, and not only when it interacts with its ligands PD-L1 or PD-L2, which are not expressed by every tumor. Indeed, the TCR-induced calcium and ERK responses were reduced in peripheral blood T cells transfected with PD-1. Inhibition by sodium stibogluconate of the SHP-1 and SHP-2 phosphatases that associate with several inhibitory receptors including PD-1, relieves part of the anergy apparent in TIL or in PD-1-transfected T cells. This work highlights some of the molecular modifications contributing to functional defects of human TIL.     

Dendritic cells infiltrating human non-small cell lung cancer are blocked at immature stage

The efficacy of immune response to control human cancer remains controversial. It is particularly debated whether and to what extent the capacity of tumor-infiltrating dendritic cells (DC) to drive immunization can be turned off by transformed cells, leading to tumor-specific tolerance rather than immunization. To address this issue, we have characterized the DC isolated from human non-small cell lung cancer (NSCLC). These biopsy specimens contained CD11c(high) myeloid DC (mDC), but also CD11c(-) plasmacytoid DC (pDC) and a third DC subset expressing intermediate level of CD11c. Compared with peripheral blood, CD11c(high) tumor-infiltrating DC (TIDC) displayed a "semi-mature" phenotype, and TLR4 or TLR8 stimulation drove them to mature partially and to secrete limited amounts of cytokines. In contrast, most tumor-infiltrating pDC were immature but underwent partial maturation after TLR7 activation, whereas TLR9 ligation triggered low secretion of IFN-alpha. CD11c(int) mDC represented approximately 25% of total DC in tumoral and peritumoral tissues and expressed low levels of costimulatory molecules contrasting with high levels of the immunoinhibitory molecule B7-H1. Finally, the poor APC function of total TIDC even after TLR stimulation and the migratory response of both tumor-infiltrating mDC and pDC toward CCL21 and SDF-1 in vitro suggested their ability to compromise the tumor-specific immune response in draining lymph nodes in vivo. Further studies will be required to establish the specific role of the three TIDC subsets in tumor immunity and to draw conclusions for the design of therapeutic strategies.