Functional measurement of local proteolytic activity in living cells of invasive and non-invasive tumors

Proteolytic cleavage of extracellular matrix (ECM) and disruption of tissue architecture are fundamental features of tumor cell invasion. The proteolytic activity is focused in close proximity to the tumor cells. Here, we describe the possibility to quantify local proteolytic activity in the microenvironment of larger cell populations by the electrical resistance breakdown assay. The assay utilizes the transepithelial electrical resistance (TEER) of an epithelial monolayer as a sensitive indicator of monolayer integrity and permeability. Local destruction of ECM by single tumor cells was demonstrated by a second assay, based on a fluorescent matrix coating on cover slides. Local digestion of the matrix results in a reduction of fluorescence. Primary cells derived from high and low grade brain tumors as well as established cell lines of malignant gliomas and non-neural tumors of different origin (melanoma, cervical carcinoma, and breast carcinoma) were compared. Differences in proteolytic activity between tumor entities were demonstrated in both assays. Primary cells of high grade gliomas and cell lines showed TEER breakdown and local matrix destruction, while low grade brain tumors lacked matrix disintegration and disruption of cell monolayers. Taken together, both assays are capable of demonstrating local proteolytic activity and thus are versatile tools for distinguishing high and low invasive tumor cells with a potential application as diagnostic and prognostic markers in clinical investigations. The advantage of the matrix digestion assay is the requirement of only very low tumor cell numbers, whereas measurement of TEER enables precise quantification of local proteolytic processes in large and even heterogeneous tumor cultures.     

Combined action of virus injection and local tumor irradiation on tumor growth in mice

The dynamics of SCCVII transplantable tumor growth in C3H/H mice was determined after local tumor irradiation and/or virus (NDV LaSota) i.p. injection. The virus applied alone significantly suppressed tumor growth, particularly until the 19th day after tumor transplantation. Local irradiation with 30 Gy resulted in tumor disappearance followed with its regrowth about 15 days later. However, if the virus was injected after the irradiation, there was no tumor growth until the end of the 31 day observation period. It should be noted that virus application prior to local irradiation did not have any additional influence on tumor growth. Thus, the pronounced efficacy of virus applied after tumor irradiation deserves attention. It is possible that the virus injected after irradiation induced a chain of cytokine production joining the action of tumor destruction induced by irradiation. This should be further studied in clarifying the approaches to combined tumor therapy with possible cell-free vaccine production.     

Route of Administration of the TLR9 Agonist CpG Critically Determines the Efficacy of Cancer Immunotherapy in Mice

Background

The TLR9 agonist CpG is increasingly applied in preclinical and clinical studies as a therapeutic modality to enhance tumor immunity. The clinical application of CpG appears, however, less successful than would be predicted from animal studies. One reason might be the different administration routes applied in most mouse studies and clinical trials. We studied whether the efficacy of CpG as an adjuvant in cancer immunotherapy is dependent on the route of CpG administration, in particular when the tumor is destructed in situ.

Methodology/Principal Findings

In situ tumor destruction techniques are minimally invasive therapeutic alternatives for the treatment of (nonresectable) solid tumors. In contrast to surgical resection, tumor destruction leads to the induction of weak but tumor-specific immunity that can be enhanced by coapplication of CpG. As in situ tumor destruction by cryosurgery creates an instant local release of antigens, we applied this model to study the efficacy of CpG to enhance antitumor immunity when administrated via different routes: peritumoral, intravenous, and subcutaneous but distant from the tumor. We show that peritumoral administration is superior in the activation of dendritic cells, induction of tumor-specific CTL, and long-lasting tumor protection. Although the intravenous and subcutaneous (at distant site) exposures are commonly used in clinical trials, they only provided partial protection or even failed to enhance antitumor responses as induced by cryosurgery alone.

Conclusions/Significance

CpG administration greatly enhances the efficacy of in situ tumor destruction techniques, provided that CpG is administered in close proximity of the released antigens. Hence, this study helps to provide directions to fully benefit from CpG as immune stimulant in a clinical setting.     

In vivo destruction of tumor tissue by cryoablation can induce inhibition of secondary tumor growth: an experimental study

BACKGROUND: Cryoablation has been used successfully for the local treatment of several cancers. Besides local destruction, a systemic antitumor response has been postulated after cryoablation of tumor tissue. In this study we evaluate the possible systemic antitumor response induced by cryodestruction of tumor tissue in two mouse tumor models. METHODS: Mice received two subcutaneously placed tumor implants (thigh and flank) of the nonimmunogenic mouse colon tumor cell line, colon 26-B. After 7 days, the thigh implant was treated by cryoablation or excision and the effect on secondary tumor growth was determined by volume measurement of the nontreated flank tumor. Cytokine (IL-1alpha and TNF-alpha) levels in plasma were measured after treatment. Similar experiments were performed in nude mice using a human melanoma cell line (MV3). Moreover, in this model the effect of cryoablation on development of spontaneous lung metastases was evaluated. RESULTS: In the colon 26-B tumor model treatment of primary tumor implants by cryoablation resulted in a significant inhibition of secondary tumor growth compared to animals treated by surgical excision (P < 0.01). Six hours after treatment, plasma levels of IL-1alpha and TNF-alpha were higher after cryoablation than after excision (P < 0.01). Also in the nude mice model cryoablation resulted in inhibition of secondary tumor growth, though not significant. Mice treated by cryoablation showed significantly less lung metastases compared to those treated by excision (P = 0.03). CONCLUSIONS: Cryoablation of tumor tissue can result in inhibition of secondary and metastatic tumor growth. A cytokine response induced by cryoablation of tumor tissue may attribute to this feature.     

Reconstruction of the lower lip after radical resection for cancer: the "Pharaoh technique".

For patients affected by far advanced lower lip cancer, with great local tumor destruction but few or no metastases, we advocate radical resection. Here we describe a method for these repairs, using two deltopectoral flaps. It produces a good functional result and satisfactory appearance. No recurrences have been observed in our 5 cases during a two-year follow-up.     

Effects of neoplasms on inflammation: depression of macrophage accumulation after tumor implantation.

The local accumulation of macrophages at sites of neoplasms may be a critical event in immunologically mediated tumor killing. Individuals with neoplasms, however, have been noted to have depressed monocyte chemotactic responsiveness in vitro. To determine the effect of neoplasms on macrophage migration, mice were implanted subcutaneously with either sarcoma or hepatoma cells and their macrophage migratory function quantified in vivo and in vitro. The ability of tumor-bearing animals to mobilize macrophages to an inflammatory site in vivo was depressed by as much as 61% by 6 days after tumor implantation. The in vitro chemotactic responsiveness of macrophages recovered from the peritoneal cavities of tumor-bearing animals was also markedly depressed. Macrophage migration was not affected by implantation of normal syngeneic or allogeneic tissues. In addition, the accumulation of polymorphonuclear leukocytes in vivo was not depressed in tumor-bearing animals. These findings suggest that neoplasms themselves may depress the host's ability to localize macrophages at inflammatory sites in vivo and thereby hinder immunologically mediated tumor destruction.     

Intratumoral injection of alpha-gal glycolipids induces xenograft-like destruction and conversion of lesions into endogenous vaccines

This study describes a novel cancer immunotherapy treatment that exploits the natural anti-Gal Ab to destroy tumor lesions and convert them into an endogenous vaccine targeted to APC via FcgammaR. Anti-Gal constitutes 1% of immunoglobulins in humans and interacts specifically with alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R). The binding of anti-Gal to alpha-gal epitopes on pig cells mediates xenograft rejection. The proposed method uses glycolipid micelles with multiple alpha-gal epitopes (alpha-gal glycolipids). These glycolipids are extracted from rabbit red cell membranes and are comprised of ceramides with carbohydrate chains containing 5-25 carbohydrates, all capped with alpha-gal epitopes. Efficacy of this treatment was demonstrated in alpha1,3-galactosyltransferase knockout mice producing anti-Gal and bearing B16 melanoma or B16/OVA producing OVA as a surrogate tumor Ag. These mice are unique among nonprimate mammals in that, similar to humans, they lack alpha-gal epitopes and can produce the anti-Gal Ab. Intratumoral injection of alpha-gal glycolipids results in local inflammation mediated by anti-Gal binding to the multiple alpha-gal epitopes and activation of complement. These glycolipids spontaneously insert into tumor cell membranes. The binding of anti-Gal to alpha-gal expressing tumor cells induces the destruction of treated lesions as in anti-Gal-mediated xenograft rejection. Anti-Gal further opsonizes tumor cells within the lesion and, thus, targets them for effective uptake by APC that transport the tumor Ags to draining lymph nodes. APC further cross-present immunogenic tumor Ag peptides and elicit a systemic anti-tumor immune response. Similar intratumoral injection of alpha-gal glycolipids in humans is likely to induce the destruction of treated lesions and elicit a protective immune response against micrometastases.     

The enter of viruses family Picornaviridae in residents macrophages

Viruses enter in cells through clathrin- and dinamin-mediated uptake route-endocytosis, caveolae-mediated local destruction of cell plasma membranes, and macropinocytosis. The non-enveloped viruses to which Picornaviridae famiy is attributed are important human and animal pathogens. The aim of this study was to examine the mechanisms of penetration of viruses of this family (polio-, echo 11-, entero 71- and coxsackie B1-viruses) into resident macrophages. After attachment to the plasma membrane of macrophages the enterovirus 71 and coxsackievirus B1 penetrated into macrophages by invagination of the plasma membrane and formation of intracytoplasmic vesicules - caveoles. The poliovirus entered macrophages both by caveols formation and local destruction of plasma membranes of the host cells. Macropinocytos of polioviruses was observed after 45 min contact. The echovirus 11 entered in host macrophages by local destruction of their plasma membranes during first 15 min. Then the formation of endocytosed vesicles with included viruses was observed. The echovirus 11 went out of endocytosed vesicles by local destruction of membrane vesicles.     

Mechanical Disruption of Tumors by Iron Particles and Magnetic Field Application Results in Increased Anti-Tumor Immune Responses

The primary tumor represents a potential source of antigens for priming immune responses for disseminated disease. Current means of debulking tumors involves the use of cytoreductive conditioning that impairs immune cells or removal by surgery. We hypothesized that activation of the immune system could occur through the localized release of tumor antigens and induction of tumor death due to physical disruption of tumor architecture and destruction of the primary tumor in situ. This was accomplished by intratumor injection of magneto-rheological fluid (MRF) consisting of iron microparticles, in Balb/c mice bearing orthotopic 4T1 breast cancer, followed by local application of a magnetic field resulting in immediate coalescence of the particles, tumor cell death, slower growth of primary tumors as well as decreased tumor progression in distant sites and metastatic spread. This treatment was associated with increased activation of DCs in the draining lymph nodes and recruitment of both DCs and CD8(+)T cells to the tumor. The particles remained within the tumor and no toxicities were observed. The immune induction observed was significantly greater compared to cryoablation. Further anti-tumor effects were observed when MRF/magnet therapy was combined with systemic low dose immunotherapy. Thus, mechanical disruption of the primary tumor with MRF/magnetic field application represents a novel means to induce systemic immune activation in cancer.     

The nature of host tissue destruction in tumor invasion. An experimental investigation on carcinoma and sarcoma xenotransplants

The nature of host tissue destruction in tumor invasion was investigated in experimentally induced carcinomas and sarcomas, xenografted into skeletal muscle. By means of light and electron microscopy it was shown that in both carcinomas and sarcomas the confrontation of host tissue with the invading tumor cells does not result in immediate destruction of host tissue but in a transitory state of coexistence which gradually proceeds to progressive host tissue atrophy. This process of progressive atrophy, which finally results in the total disappearance of the invaded host tissue, is considered to be caused mainly by the increasing pressure and competitive withdrawal of oxygen and nutrients by the invading and proliferating tumor cells. Morphological changes suggesting an active enzymatic breakdown of host tissue cells by tumor cells were not observed during any stage of tumor invasion.     

Destruction of tumor cells by BCG-activated alqolar macrophages.

Alveolar macrophages obtained from Syrian golden hamsters were tested for their ability to destroy tumor cells. Only macrophages obtained from BCG immune animals rechallenged intratracheally with BCG five days before assay exhibited cytotoxic activity. Maximum destruction of tumor cells occurred after 5 days of incubation. Immunologic activation of macrophages was required to attain cytotoxic alveolar macrophages. Induction of inflammatory lung exudates by a variety of nonspecific irritants did not result in tumor cell destruction by macrophages. These observations may prove useful in designing an approach for immunotherapy of lung cancer.     

Cryosurgery of pulmonary metastases.

Indications and results of 33 cryosurgical interventions for metastatic tumors in the lung are presented. Regression of local and metastatic pulmonary growth on the contralateral side was observed in four cases. Nine cases showed temporary halt of metastatic pulmonary tumor growth. The technique of cryosurgery for pulmonary metastases is reviewed. The procedure of cryosurgery of pulmonary metastases was found to be an innocuous method to attempt both tumor destruction and eventually specific immunologic stimulation. Preliminary observations with the lymphocytes and sera indicate that cryosurgery of pulmonary metastases induces an increase in specific cell mediated immune response without producing blocking serum factors and may give rise to specific, complement dependent cytotoxic antibodies. In one case both mechanisms were observed after cryotherapy. In three cases with progressive disease, lymphocyte mediated cytotoxicity alone was stimulated.     

Modulation of the "blood-tumor" barrier improves immunotherapy

Blood vessels inside tumors are crucial for cancer survival and progression but equally contribute to the tumor's intrinsic resistance to therapy. Abnormal blood flow in the local tumor environment acts as a physiological barrier to the delivery of chemotherapeutic agents. Furthermore, tumor vasculature can also act as a barrier for immune cell migration into the tumor parenchyma. Much has been made of anti-angiogenic therapies that specifically inhibit vessel growth. However, recent findings demonstrate that the chaotic architecture of tumor blood vessels can be reversed which in turn normalizes blood flow and physical parameters in the tumor environment. Importantly, vessel normalization also improves lymphocyte migration into tumor tissue and immune destruction. Identification of regulator of G protein signaling 5 (RGS5) as a key modulator of the vascular barrier in tumor progression and regression has brought new insights into the molecular basis of vessel normalization and opens new therapeutic opportunities.     

Pre-conditioning cryosurgery: cellular and molecular mechanisms and dynamics of TNF-α enhanced cryotherapy in an in vivo prostate cancer model system

Cryosurgery is increasingly being used to treat prostate cancer; however, a major limitation is local recurrence of disease within the previously frozen tissue. We have recently demonstrated that tumor necrosis factor alpha (TNF-α), given 4h prior to cryosurgery can yield complete destruction of prostate cancer within a cryosurgical iceball. The present work continues the investigation of the cellular and molecular mechanisms and dynamics of TNF-α enhancement on cryosurgery. In vivo prostate tumor (LNCaP Pro 5) was grown in a dorsal skin fold chamber (DSFC) on a male nude mouse. Intravital imaging, thermography, and post-sacrifice histology and immunohistochemistry were used to assess iceball location and the ensuing biological effects after cryosurgery with and without TNF-α pre-treatment. Destruction was specifically measured by vascular stasis and by the size of histologic zones of injury (i.e., inflammatory infiltrate and necrosis). TNF-α induced vascular pre-conditioning events that peaked at 4h and diminished over several days. Early events (4-24 h) include upregulation of inflammatory markers (nuclear factor-κB (NFκB) and vascular cell adhesion molecule-1 (VCAM)) and caspase activity in the tumor prior to cryosurgery. TNF-α pre-conditioning resulted in recruitment of an augmented inflammatory infiltrate at day 3 post treatment vs. cryosurgery alone. Finally, pre-conditioning yielded enhanced cryosurgical destruction up to the iceball edge at days 1 and 3 vs. cryosurgery alone. Thus, TNF-α pre-conditioning enhances cryosurgical lesions by vascular mechanisms that lead to tumor cell injury via promotion of inflammation and leukocyte (esp. neutrophil) recruitment.     

Overcoming immune evasion in T cell therapy of cancer: lessons from animal models

Cancer antigen-specific cytotoxic T lymphocytes (CTL) are the major effectors against cancer cells. However, large established tumors are usually not fully controlled by CTL for at least two reasons. First, large established tumors have immune suppressive networks that not only suppress CTL effector function but also permit tumor progression. Second, the genetic instability of cancer cells often results in the selection of antigenic variants by CTL, which allow cancer cells to escape destruction. Simply enhancing T cell capacity may not fully control large established tumors. Other measures, such as enhancing local costimulation, inhibiting angiogenesis and down-regulating functions of tumor associated myeloid cells should also be considered. In this paper we will review some of the progress from animal studies.     

Pre-conditioning cryosurgery: Cellular and molecular mechanisms and dynamics of TNF-α enhanced cryotherapy in an in vivo prostate cancer model system

Cryosurgery is increasingly being used to treat prostate cancer; however, a major limitation is local recurrence of disease within the previously frozen tissue. We have recently demonstrated that tumor necrosis factor alpha (TNF-α), given 4 h prior to cryosurgery can yield complete destruction of prostate cancer within a cryosurgical iceball. The present work continues the investigation of the cellular and molecular mechanisms and dynamics of TNF-α enhancement on cryosurgery. In vivo prostate tumor (LNCaP Pro 5) was grown in a dorsal skin fold chamber (DSFC) on a male nude mouse. Intravital imaging, thermography, and post-sacrifice histology and immunohistochemistry were used to assess iceball location and the ensuing biological effects after cryosurgery with and without TNF-α pre-treatment. Destruction was specifically measured by vascular stasis and by the size of histologic zones of injury (i.e., inflammatory infiltrate and necrosis). TNF-α induced vascular pre-conditioning events that peaked at 4 h and diminished over several days. Early events (4–24 h) include upregulation of inflammatory markers (nuclear factor-κB (NFκB) and vascular cell adhesion molecule-1 (VCAM)) and caspase activity in the tumor prior to cryosurgery. TNF-α pre-conditioning resulted in recruitment of an augmented inflammatory infiltrate at day 3 post treatment vs. cryosurgery alone. Finally, pre-conditioning yielded enhanced cryosurgical destruction up to the iceball edge at days 1 and 3 vs. cryosurgery alone. Thus, TNF-α pre-conditioning enhances cryosurgical lesions by vascular mechanisms that lead to tumor cell injury via promotion of inflammation and leukocyte (esp. neutrophil) recruitment.     

Remission of Collagen-Induced Arthritis through Combination Therapy of Microfracture and Transplantation of Thermogel-Encapsulated Bone Marrow Mesenchymal Stem Cells

The persistent inflammation of rheumatoid arthritis (RA) always leads to partial synovial hyperplasia and the destruction of articular cartilage. Bone marrow mesenchymal stem cells (BMMSCs) have been proven to possess immunosuppressive effects, and widely explored in the treatment of autoimmune diseases. However, poor inhibitory effect on local inflammatory state and limited capacity of preventing destruction of articular cartilage by systemic BMMSCs transplantation were observed. Herein, toward the classical type II collagen-induced arthritis in rats, the combination treatment of microfracture and in situ transplantation of thermogel-encapsulated BMMSCs was verified to obviously down-regulate the ratio of CD4⁺ to CD8⁺ T lymphocytes in peripheral blood. In addition, it resulted in the decreased levels of inflammatory cytokines, such as interleukin-1β, tumor necrosis factor-α and anti-collagen type II antibody, in the serum. Simultaneously, the combination therapy also could inhibit the proliferation of antigen specific lymphocytes and local joint inflammatory condition, and prevent the articular cartilage damage. The results indicated that the treatment programs could effectively stimulate the endogenous and exogenous BMMSCs to exhibit the immunosuppression and cartilage protection capability. This study provided a new therapeutic strategy for autoimmune inflammatory diseases, such as RA.     

Depressed Immune Surveillance Against Cancer: Role of Deficient T Cell: Extracellular Matrix Interactions

Although T cells infiltrate malignant tumors, the local immune response is usually inefficient and tumors escape destruction. While extracellular matrix proteins strongly costimulate T cell responses in normal individuals, our studies indicate that peripheral blood T cells from cancer patients and tumor infiltrating cells respond poorly or are resistant to stimulative signals mediated by collagen I and IV and fibronectin. Moreover, the adhesive properties of cancer T cells are markedly depressed. Those functional deficiencies are paralleled by variable deficits in integrin and non-integrin T cell receptors for extracellular matrix. Immunotherapy with BCG causes a dramatic but transient increase in T cell: ECM interactions.     

Depressed Immune Surveillance Against Cancer: Role of Deficient T Cell: Extracellular Matrix Interactions

Although T cells infiltrate malignant tumors, the local immune response is usually inefficient and tumors escape destruction. While extracellular matrix proteins strongly costimulate T cell responses in normal individuals, our studies indicate that peripheral blood T cells from cancer patients and tumor infiltrating cells respond poorly or are resistant to stimulative signals mediated by collagen I and IV and fibronectin. Moreover, the adhesive properties of cancer T cells are markedly depressed. Those functional deficiencies are paralleled by variable deficits in integrin and non-integrin T cell receptors for extracellular matrix. Immunotherapy with BCG causes a dramatic but transient increase in T cell: ECM interactions.     

Efferent connections of the caudate nucleus in cats

Structural and ultrastructural changes in the frontal areas of the cortex and in the region of the globus pallidus were investigated after local and extensive destruction of the caudate nucleus. It was shown by the Fink-Heimer method that after local injury to the caudate nucleus by means of electrodes implanted 2–16 months before electrolytic destruction, only a few degenerating fibers of medium and thin caliber were present. Extensive destruction of the caudate nucleus (without preimplantation of electrodes) was followed by massive degeneration of fibers of different caliber in the frontal area of the cortex. After local injury to the caudate nucleus numerous thin degenerating axons 0.5–0.6 µ in diameter and degenerating terminals were found in the region of the globus pallidus. Degenerative changes in the axo-dendritic and axo-somatic terminals followed the "dark" type of course. It is concluded that no considerable direct projections of neurons of the caudate nucleus are present in the cortex. Degenerating fibers of average caliber in frontal areas of the cortex after destruction of the caudate nucleus are evidently axons of thalamic neurons and not from cells of the damaged nucleus.A. A. Bogomol'ets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 7, No. 2, pp. 165–171, March–April, 1975.