Clonal analysis of a transplantable rat seminoma]

The study of rat testis seminoma by the method of clonal analysis and ectopic transplantations has shown that typical and anaplastic types are different stages of tumor progression in the course of which atypical and anaplastic features of the seminoma cell elements increase. It does not seem inconceivable that the spermacytic form, never encountered during our investigation of 400 tumors, may be an independent seminoma form incapable of transforming into a typical one. A spindle-cell form of seminoma has been described most frequently occurring during transplantation of a typical seminoma into testis and during cloning. The obtained evidence permits to consider the small dark cells of the seminoma as being cambial cells of the tumor which occur abundantly in growing tumors, and are absent in tumors with ceased proloferating. At later stages of the seminoma development, the small dark cells may be absent, their function being performed practically by all tumor cells. Acinar structures found in part of clones from lungs and in the majority of transplants from eye anterior chamber suggest a histogenetic relationship between the typical seminoma and embryocarcinoma, and thus enable us to regard the latter as the most differentiated variants of the seminoma.     

CTL induction of tumoricidal nitric oxide production by intratumoral macrophages is critical for tumor elimination

To characterize mechanisms of CTL inhibition within an ocular tumor microenvironment, tumor-specific CTLs were transferred into mice with tumors developing within the anterior chamber of the eye or skin. Ocular tumors were resistant to CTL transfer therapy whereas skin tumors were sensitive. CTLs infiltrated ocular tumors at higher CTL/tumor ratios than in skin tumors and demonstrated comparable ex vivo effector function to CTLs within skin tumors indicating that ocular tumor progression was not due to decreased CTL accumulation or inhibited CTL function within the eye. CD11b(+)Gr-1(+)F4/80(-) cells predominated within ocular tumors, whereas skin tumors were primarily infiltrated by CD11b(+)Gr-1(-)F4/80(+) macrophages (Ms), suggesting that myeloid derived suppressor cells may contribute to ocular tumor growth. However, CD11b(+) myeloid cells isolated from either tumor site suppressed CTL activity in vitro via NO production. Paradoxically, the regression of skin tumors by CTL transfer therapy required NO production by intratumoral Ms indicating that NO-producing intratumoral myeloid cells did not suppress the effector phase of CTL. Upon CTL transfer, tumoricidal concentrations of NO were only produced by skin tumor-associated Ms though ocular tumor-associated Ms demonstrated comparable expression of inducible NO synthase protein suggesting that NO synthase enzymatic activity was compromised within the eye. Correspondingly, in vitro-activated Ms limited tumor growth when co-injected with tumor cells in the skin but not in the eye. In conclusion, the decreased capacity of Ms to produce NO within the ocular microenvironment limits CTL tumoricidal activity allowing ocular tumors to progress.     

Detection of the range of cytological changes in tumors induced in rats by nitrosoamines by means of passage in the anterior chamber of the eye]

12 rat liver tumors (high, purely and low differentiated hepatomas and adenocarcinomas), 3 kidney tumors, 1 lymphosarcoma and 1 round cell sarcoma of the uterus were transplanted and passed trough anterior chamber of the allogenic rat eyes. In 8 primary transplants of the liver tumors differentiation traits increased; three of them were morphologically undistinguishable from the normal liver tissue. In some transplants of embryonic liver and kidney displasia was detected. The results obtained give evidence in favor of a very high cytological variability of tumors at the early steps of progression, and indicate that the cultivation in the anterior chamber of the eye may be a proper model for investigation of cytological variability of both malignant and normal tissues.     

Rejection of intraocular tumors by CD4(+) T cells without induction of phthisis.

Immune privilege of the eye protects against sight-threatening inflammatory events, but can also permit outgrowth of otherwise nonlethal immunogenic tumors. Nonetheless, ocular tumor growth can be controlled by cellular immune responses. However, this will normally result in phthisis of the eye, in case tumor rejection is mediated by a delayed-type hypersensitivity response orchestrated by CD4(+) T cells. We now show that intraocular tumors can be eradicated by CD4(+) Th cells without inducing collateral damage of neighboring ocular tissue. Injection of tumor cells transformed by the early region 1 of human adenovirus type 5 in the anterior chamber of the eye leads to intraocular tumor formation. Tumor growth is transient in immunocompetent mice, but lethal in immunodeficient nude mice, indicating that T cell-dependent immunity is responsible for tumor clearance. Tumor rejection has all the characteristics of a CD8(+) T cell-mediated immune response, as the tumor did not express MHC class II and only tumor tissue was the subject of destruction. However, analysis of the molecular and cellular mechanisms involved in tumor clearance revealed that perforin, TNF-alpha, Fas ligand, MHC class I, and CD8(+) T cells did not play a crucial role in tumor eradication. Instead, effective tumor rejection was entirely dependent on CD4(+) Th cells, as CD4-depleted as well as MHC class II-deficient mice were unable to reject their intraocular tumor. Taken together, these observations demonstrate that CD4(+) T cells are able to eradicate MHC class II-negative tumors in an immune-privileged site without affecting surrounding tissues or the induction of phthisis.     

Immunogenetic basis for immunologic privilege in the anterior chamber of the eye

DBA/2 mastocytoma (P815) cells are able to grow when inoculated into the anterior chamber of eyes of histoincompatible mice. Tumor growth is unrestrained in recipient mice differing from the DBA/2 strain at multiple minor H loci, but sharing the H-2 ^d haplotype; progressive tumor growth in these animals involves the entire eye, invades the orbit and kills the hosts by extension into the cranial vault. Alternatively, P815 cells grow initially but are unable to sustain continued growth in the anterior chambers of recipient mice differing from DBA/2 at the H-2 complex. Recipients differing from DBA/2 at either the K or D regions of H-2 develop anti-DBA/2 immunity that destroys the intraocular tumor within 20 days of inoculation. Severe inflammatory reactions in these eyes produce innocent bystander destruction of ocular tissue and produce blindness. Recipients differing from DBA/2 at both K and D regions of H-2 also mount vigorous anti-DBA/2 immunity that destroys the intraocular tumor. In these instances, the nonspecific component of the rejection reaction is minimal: when the tumor cells are destroyed, the eyes are restored to anatomic and functional integrity. These results indicate that immunologic privilege in the anterior chamber of the eye is afforded to allogeneic tissues to varing degrees depending upon allodisparity at H-2 loci encoding class I MHC products. The results further imply that the precision of the alloimmune response may be under the control of these same MHC products.     

Tumor escape mutants develop within an immune-privileged environment in the absence of T cell selection

The establishment of tumor escape mutants, which can be driven by innate and/or adaptive immune effector cells, presents a significant obstacle in the development of successful tumor immunotherapies. Our study documents that tumors growing within an immune-privileged site within the eye develop a tumor escape phenotype in the absence of selective T cell pressure. P815 tumor cells that are recovered from progressively growing tumors within the anterior chamber of the eye escape elimination when injected into the flanks of a second group of syngeneic DBA/2 mice that were previously immunized against P815 tumor cells. The escape phenotype of eye-derived P815 tumors was stable and permanent when the tumor cells were cultured in vitro. Eye-derived tumor cells recovered from the anterior chamber of CB-17 SCID mice also escaped elimination when injected into the flanks of immunized mice, demonstrating that selective pressure by tumor Ag-specific T cells did not contribute to the development of the escape phenotype. In vitro studies demonstrated that eye-derived tumor cells were not lysed by specific CTL and were unable to restimulate primed Ag-specific T cells. Immune escape of eye-derived tumor cells was not due to down-regulation of either MHC class I or ICAM-1. Our data demonstrate that the immune-privileged environment within the eye induces a tumor escape phenotype that is not driven by selective T cell pressure. We predict that immune escape within the eye is driven by the unique ocular environment that permanently alters gene expression in eye-derived tumor cells.     

Capacity for differentiation and normalization of populations of tumor cells transplanted into the anterior chamber of the eye. I. Changes in the pleomorphic rhabdomyosarcomas MKh-III and A-7 of mice

Tumors transplanted into the eye anterior chamber (EAC) were atypic, more differentiated morphologically and with higher activity of H-forms of LDH, compared to the same tumors grown in the subcutaneous connective tissue (SCT). Besides, in the EAC, the tumors were less karyotypically heterogeneous and had lesser mitotic indices, than in the SCT. After cultivation in the EAC, populations of tumor rhabdomyoblasts displayed a decreased transplantability in SCT, i.e. the usual site of their implantation. These results may suggest that the proliferation of tumor cell populations in the EAC, i.e. in the immunologically privileged place, can be accompanied by the reducing of their malignancy up to the total loss of the common tumorigenic ability. Even the cell populations of tumor rhabdomyoblasts from the late stages of tumor progression can undergo such a process of normalization.     

Noninvasive high-resolution in vivo imaging of cell biology in the anterior chamber of the mouse eye

There is clearly a demand for an experimental platform that enables cell biology to be studied in intact vascularized and innervated tissue in vivo. This platform should allow observations of cells noninvasively and longitudinally at single-cell resolution. For this purpose, we use the anterior chamber of the mouse eye in combination with laser scanning microscopy (LSM). Tissue transplanted to the anterior chamber of the eye is rapidly vascularized, innervated and regains function. After transplantation, LSM through the cornea allows repetitive and noninvasive in vivo imaging at cellular resolution. Morphology, vascularization, cell function and cell survival are monitored longitudinally using fluorescent proteins and dyes. We have used this system to study pancreatic islets, but the platform can easily be adapted for studying a variety of tissues and additional biological parameters. Transplantation to the anterior chamber of the eye takes 25 min, and in vivo imaging 1-5 h, depending on the features monitored.     

Accumulation of immunosuppressive CD11b+ myeloid cells correlates with the failure to prevent tumor growth in the anterior chamber of the eye

The purpose of these studies is to determine why an immunogenic tumor grows unchecked in the anterior chamber (a.c.) of the eye. The OVA-expressing EL4 tumor, E.G7-OVA, was injected into the a.c. or skin of immunocompetent and immunodeficient mice. Tumor growth and tumor-specific immune responses were monitored. Ocular tumor-infiltrating leukocytes were characterized phenotypically and functionally. Growth of E.G7-OVA was inhibited when limiting numbers of cells were injected in the skin but not in the a.c. of C57BL/6 mice, although both routes primed OVA-specific immune responses, which prevented the growth of a subsequent injection with E.G7-OVA in the skin or opposite eye. Tumor regression was OVA-specific because growth of the parental EL-4 tumor was not inhibited in primed mice. E.G7-OVA growth in the skin was not inhibited in immunodeficient Rag(-/-) or CD8 T cell-deficient mice, suggesting that CD8(+) CTLs mediate tumor elimination. CD8(+) T cell numbers were significantly increased in eyes of mice primed with E.G7-OVA, but few were detected in primary ocular tumors. Nevertheless, growth of E.G7-OVA was retarded in the a.c. of TCR-transgenic OT-I mice, and CD8(+) T cell numbers were increased within eyes, suggesting that tumor-specific CD8(+) CTLs migrated into and controlled primary ocular tumor growth. E.G7-OVA did not lose antigenicity or become immunosuppressive after 13 days of growth in the eye. However, CD11b(+) cells accumulated in primary ocular tumors and contained potent immunosuppressive activity when assayed in vitro. Thus, CD11b(+) cells that accumulate within the eye as tumors develop in the a.c. may contribute to immune evasion by primary ocular tumors by inhibiting CTLs within the eye.     

Cell populations of transplantable murine tumors of various degrees of histogenesis during their proliferation in the anterior chamber

Six transplantable murine tumors of different histogenesis were investigated after transplantation to subcutaneous connective tissue (SCT) and the eye anterior chamber (EAC). Cell morphology was studied using light microscopy. DNA contents in the nuclei of tumor cells were investigated with flow cytometry technique. LDH isoenzymes were studied using electrophoresis in polyacrylamide gel. In the case of tumors with near diploid modal class, a redistribution of LDH isoenzyme activity and an increase in morphological differentiation level were obtained. In the case of tumors with modal class differing from the diploid one, a morphological structure changes were revealed, but there were no differences in LDH isoenzyme activity. The data obtained show that the capability of increasing morphological and biochemical differentiation level after cultivation in the EAC of murine transplantable tumors remains even on the late stages of progression in tumors of different histogenesis with near diploid value of modal class.     

Hypoxia as pathogenic factor affecting the eye tissues: The selective apoptotic damage of the conjunctiva and anterior epithelium of the cornea

The effect of acute hypoxia on the occurrence of apoptosis in eye cells in rats placed in a pressure chamber was studied. Selective primary lesion of cells of the conjunctiva and the anterior corneal epithelium was found. A possible role of the simulated hypoxic conditions in the dry eye syndrome pathogenesis, which is accompanied by primary lesion of cells in the anterior eye surface tissues is discussed.     

Ligand-independent activation of the Hedgehog pathway displays non-cell autonomous proliferation during eye development in Drosophila

Deregulation of the Hedgehog (Hh) signaling pathway is associated with the development of human cancer including medullobastoma and basal cell carcinoma. Loss of Patched or activation of Smoothened in mouse models increases the occurrence of tumors. Likewise, in a Drosophila eye model, deregulated Hedgehog signaling causes overgrowth of eye and head tissues. Surprisingly, we show that cells with deregulated Hh signaling do not or only little contribute to the tissue overgrowth. Instead, they become more sensitive to apoptosis and may eventually be eliminated. Nevertheless, these mutant cells increase proliferation in the adjacent wild-type tissue, i.e., in a non-cell autonomous manner. This non-cell autonomous effect is position-dependent and restricted to mutant cells in the anterior portion of the eye. We also observe precocious non-cell autonomous differentiation in genetic mosaics with deregulated Hh signaling. Together, these non-cell autonomous growth and differentiation phenotypes in the Drosophila eye model reveal another strategy by which oncogenes may generate a supportive micro-environment for tumor growth.     

Motility cues in the tumor microenvironment

It is now increasingly recognized that the microenvironment plays a critical role in the progression of tumors. Perhaps less obvious is the concept that the microenvironment may share responsibility in determining the "malignant" traits of tumor cells, i.e. invasiveness and metastasis. If tumors are tissues, however unbalanced, rather than a collection of "malignant" cells recruiting local resources for the purpose of growth, then it is inevitable that tumor cells will respond to local stimuli. These stimuli include cues for motility and migration, which normally appear in tissues undergoing formation, remodeling or healing. Carcinoma cells are likely to be sensitive to the motility cues that normally regulate epithelial morphogenetic movements such as ingression, delamination, invagination, and tube or sheet migration. "Malignant" tumors, then, can be redefined as those in which these cues arise more frequently or act more effectively. Here, we expand on this view and propose that invasion and metastasis may be the outcome of tumor cell responses to microenvironmental motility cues. Understanding how such motility cues arise and act, both in normal and tumor tissue, should be a high priority in cancer research.     

Experimental Boron Neutron Capture Therapy for Melanoma: Systemic Delivery of Boron to Melanotic and Amelanotic Melanoma

The boron-containing melanin precursor analogue p-boronophenylalanine (BPA) has previously been shown to selectively deliver boron to pigmented murine melanomas when administered in a single intragastric dose. If boron neutron capture therapy is to become a clinically useful method of radiation therapy for human malignant melanoma, the boron carrier must be capable of delivering useful amounts of boron to remote tumor sites (metastases) and to poorly pigmented melanomas. We have now determined the ability of BPA to accumulate in several nonpigmented melanoma models including human melanoma xenografts in nude mice. The absolute amount of boron in the nonpigmented melanomas was about 50% of that observed in the pigmented counterparts but was still selectively concentrated in the tumor relative to normal tissues in amounts sufficient for effective neutron capture therapy. Single intragastric doses of BPA resulted in selective localization of boron in the amelanotic Greene melanoma carried in the anterior chamber of the rabbit eye and in a pigmented murine melanoma growing in the lungs. The ratio of the boron concentration in these tumors to the boron concentration in the immediately adjacent normal tissue was in the range of 3:1 to 4:1. These distribution studies support the proposal that boron neutron capture therapy may be useful as a regional therapy for malignant melanoma.     

Role of TRAIL and IFN-gamma in CD4+ T cell-dependent tumor rejection in the anterior chamber of the eye

Although the anterior chamber of the eye expresses immune privilege, some ocular tumors succumb to immune rejection. Previous studies demonstrated that adenovirus-induced tumors, adenovirus type 5 early region 1 (Ad5E1), underwent immune rejection following transplantation into the anterior chamber of syngeneic mice. Intraocular tumor rejection required CD4(+) T cells, but did not require the following: 1) CD8(+) T cells, 2) B cells, 3) TNF, 4) perforin, 5) Fas ligand, or 6) NK cells. This study demonstrates that CD4(+) T cell-dependent tumor rejection does not occur in IFN-gamma-deficient mice. Ad5E1 tumor cells expressed DR5 receptor for TRAIL and were susceptible to TRAIL-induced apoptosis. Although IFN-gamma did not directly induce apoptosis of the tumor cells, it rendered them 3-fold more susceptible to TRAIL-induced apoptosis. Both CD4(+) T cells and corneal endothelial cells expressed TRAIL and induced apoptosis of Ad5E1 tumor cells. The results suggest that Ad5E1 tumor rejection occurs via TRAIL-induced apoptosis as follows: 1) tumor cells express TRAIL-R2 and are susceptible to TRAIL-induced apoptosis, 2) IFN-gamma enhances TRAIL expression on CD4(+) T cells and ocular cells, 3) IFN-gamma enhances tumor cell susceptibility to TRAIL-induced apoptosis, 4) apoptotic tumor cells are found in the eyes of rejector mice, but not in the eyes of IFN-gamma knockout mice that fail to reject intraocular tumors, 5) CD4(+) T cells and corneal endothelial cells express TRAIL and induce apoptosis of tumor cells, and 6) apoptosis induced by either CD4(+) T cells or corneal cells can be blocked with anti-TRAIL Ab.     

Intraocular tumor antigen drains specifically to submandibular lymph nodes, resulting in an abortive cytotoxic T cell reaction

Ocular immune privilege is considered essential in the protection against sight-threatening immune responses, as illustrated by the ability of the ocular environment to permit the growth of tumors that are rejected when implanted at other sites. Although several studies indicate that soluble Ag can drain directly into the spleen when injected into the anterior chamber, the primary site of intraocular tumor Ag presentation to tumor-specific CTLs has not been studied. To gain a better understanding of the mechanism involved in ocular immune privilege, we examined to which lymphoid organs anterior chamber tumor Ags primarily drain. Our data show that intraocular tumor Ag drains exclusively to the submandibular lymph nodes, resulting in activation of tumor-specific CTLs, whereas no Ag drainage was found in spleen. However, these tumor-specific CTLs do not distribute systemically and, as a consequence, intraocular tumor growth is unhampered. A similar lack of CTL efficacy has been observed in mice bearing s.c. tumors, which is converted to a systemic tumoricidal CTL response by administration of agonistic anti-CD40 mAb. In contrast, systemic anti-CD40 treatment of eye tumor-bearing mice did not result in mobilizing tumor-specific CTLs or tumor eradication. Together, these results show that intraocular tumor Ag drains to regional lymph nodes for activation of tumor-specific CTLs. However, the induced tumor-specific immunity is insufficient for tumor clearance, even combined with otherwise highly effective immune intervention protocols.     

Maintenance of immune tolerance depends on normal tissue homeostasis

Ags expressed at immune privileged sites and other peripheral tissues are able to induce T cell tolerance. In this study, we analyzed whether tolerance toward an intraocular tumor expressing a highly immunogenic CTL epitope is maintained, broken, or reverted into immunity in the event the anatomical integrity of the eye is lost. Inoculation of tumor cells into the anterior chamber of the eye of naive B6 mice leads to progressive intraocular tumor growth, an abortive form of CTL activation in the tumor-draining submandibular lymph node, and systemic tolerance as evidenced by the inability of these mice to reject an otherwise benign tumor cell inoculum. Loss of anatomical integrity of the eye as a consequence of phthisis resulted in loss of systemic tolerance and the emergence of effective antitumor immunity against an otherwise lethal tumor challenge. Phthisis was accompanied by dendritic cell maturation and preceded the induction of systemic tumor-specific CTL immunity. Our data show that normal tissue homeostasis and anatomical integrity is required for the maintenance of ocular tolerance and prevention of CTL-mediated immunity. These data also indicate that tissue injury in the absence of viral or microbial infection can act as a switch for the induction of CTL immunity.     

The histology of pigmented Bomirski melanoma growing in the hamster eye--preliminary results

We demonstrated that a quickly growing hamster skin melanoma developed a tumor after autologous implantation into the anterior chamber of the eye. Tumor cells were seen invading all the surrounding tissues, including the iris, ciliary body, choroid and cornea. Histological examination confirmed the presence of numerous blood vessels of large diameter. Their walls were very thin, thus only the endothelium could be identified using light microscopy. Macrophages, microemboli and extravasations were present within the tumor mass.     

Local T helper cell signals by lymphocytes infiltrating intraocular tumors.

Tumor-infiltrating lymphocytes from mice bearing minor histoincompatible tumor cells in the anterior chamber (AC) or subconjunctival (SCon) space of the eye have been shown to contain large numbers of tumor-specific precursor cytotoxic T cells. Because SCon tumors eventually acquire directly cytotoxic, tumor-specific T cells and are rejected by their hosts and because AC tumors never acquire cytotoxic effector cells and are not rejected, we have examined tumor-infiltrating lymphocytes from both types of ocular tumors for the capacity to secrete lymphokines in response to in vitro stimulation with tumor cells. The results indicate that T "helper" cells were able to infiltrate both SCon and AC tumors. In the former, T cells capable of secreting IL-2 and IL-4 were found whereas in the latter only IL-2-secreting T cells were detected. These findings implicate a defect in local delivery of appropriate T cell help as the reason why AC tumors are not rejected. The failure of AC tumor-bearing mice to destroy their tumors correlates not only with defective delivery of local help but with a systemic inability to produce tumor-specific T cells that can secrete IL-2 and IL-4. Because these mice also generate down-regulatory T cells that suppress the expression of tumor-specific delayed hypersensitivity, they appear to have an immunologically mediated block in T helper cell differentiation which renders them unable to generate either T helper 1 or T helper 2 cells. This immunologic abnormality is discussed in terms of tumor rejection and the phenomenon of immunologic privilege.