Inhibition of hyperprogressive cancer disease induced by immune-checkpoint blockade upon co-treatment with meta-tyrosine and p38 pathway inhibitor.

BACKGROUND: Although immune-checkpoint inhibitors (ICI) are overall promissory for cancer treatment, they entail, in some cases, an undesired side-effect called hyperprogressive-cancer disease (HPD) associated with acceleration of tumor growth and shortened survival. METHODS: To understand the mechanisms of HPD we assayed the ICI therapy on two murine tumors widely different regarding immunogenicity and, subsequently, on models of local recurrences and metastases of these tumors. To potentiate the immune response (IR), we combined ICI with meta-tyrosine-that counteracts immune-suppressive signals-and a selective inhibitor of p38 pathway that proved to counteract the phenomenon of tumor-immunostimulation. RESULTS: ICI were therapeutically effective against both tumor models (proportionally to their immunogenicity) but only when they faced incipient tumors. In contrast, ICI produced acceleration of large and residual tumors. The combined treatment strongly inhibited the growth of large tumors and it managed to cure 80% of mice with local recurrences and 60% of mice bearing residual metastases. CONCLUSIONS: Tumor enhancement was paradoxically correlated to a weak increase of the antitumor IR suggesting that a weak IR - different from a strong tumor-inhibitory one-may produce stimulation of tumor growth, mimicking the HPD observed in some clinical settings.

Meta-tyrosine modulates the immune response induced by bacterial endotoxins.

Sepsis is characterized by an early pro-inflammatory phase followed by compensatory anti-inflammatory mechanisms that lead to a late generalized immunosuppression, period where most deaths occur. Immunotherapy approaches to recover the immunocompetence in sepsis are similar to those used in cancer. Meta-tyrosine (m-Tyr) is a product of oxidative stress present in circulation during the sepsis and cancer-associated pro-inflammatory stages. In this work, considering its potential participation in pro-inflammatory processes, we evaluate the effect of m-Tyr during LPS induced immunosuppression phase in a murine model. In addition, we examine the effect of m-Tyr in a vaccination strategy using a weakly immunogenic tumor model. Our results showed that m-Tyr could prevent the establishment of immunosuppression and rescue the host from an installed immunosuppression induced by LPS. These effects were parallel to the ability of m-Tyr to improve the pro-inflammatory effects induced by LPS and inhibit the anti-inflammatory action of dexamethasone. Also, m-Tyr treatment prevents both the reduction of splenic lymphocytes and the increase of the expression of programmed death ligand-1 in splenic myeloid cells associated with immunosuppression. Besides, treatment with m-Tyr increased the protective effect of an anti-tumor vaccine, suggesting that m-Tyr could improve the immune response. In summary, we suggest that m-Tyr can modulate critical immunological indicators through the inflammatory context, which could improve the management of diseases, such as sepsis and cancer, in which immunosuppression is a significant clinical problem.

Improvement of Antitumor Therapies Based on Vaccines and Immune-Checkpoint Inhibitors by Counteracting Tumor-Immunostimulation.

Immune-checkpoint inhibitors and antitumor vaccines may produce both tumor-inhibitory and tumor-stimulatory effects on growing tumors depending on the stage of tumor growth at which treatment is initiated. These paradoxical results are not necessarily incompatible with current tumor immunology but they might better be explained assuming the involvement of the phenomenon of tumor immunostimulation. This phenomenon was originally postulated on the basis that the immune response (IR) evoked in Winn tests by strong chemical murine tumors was not linear but biphasic, with strong IR producing inhibition and weak IR inducing stimulation of tumor growth. Herein, we extended those former observations to weak spontaneous murine tumors growing in pre-immunized, immune-competent and immune-depressed mice. Furthermore, we demonstrated that the interaction of specifical T cells and target tumor cells at low stimulatory ratios enhanced the production of chemokines aimed to recruit macrophages at the tumor site, which, upon activation of toll-like receptor 4 and p38 signaling pathways, would recruit and activate more macrophages and other inflammatory cells which would produce growth-stimulating signals leading to an accelerated tumor growth. On this basis, the paradoxical effects achieved by immunological therapies on growing tumors could be explained depending upon where the therapy-induced IR stands on the biphasic IR curve at each stage of tumor growth. At stages where tumor growth was enhanced (medium and large-sized tumors), counteraction of the tumor-immunostimulatory effect with anti-inflammatory strategies or, more efficiently, with selective inhibitors of p38 signaling pathways enabled the otherwise tumor-promoting immunological strategies to produce significant inhibition of tumor growth.

Game-changing restraint of Ros-damaged phenylalanine, upon tumor metastasis.

An abrupt increase in metastatic growth as a consequence of the removal of primary tumors suggests that the concomitant resistance (CR) phenomenon might occur in human cancer. CR occurs in murine tumors and ROS-damaged phenylalanine, meta-tyrosine (m-Tyr), was proposed as the serum anti-tumor factor primarily responsible for CR. Herein, we demonstrate for the first time that CR happens in different experimental human solid tumors (prostate, lung anaplastic, and nasopharyngeal carcinoma). Moreover, m-Tyr was detected in the serum of mice bearing prostate cancer (PCa) xenografts. Primary tumor growth was inhibited in animals injected with m-Tyr. Further, the CR phenomenon was reversed when secondary implants were injected into mice with phenylalanine (Phe), a protective amino acid highly present in primary tumors. PCa cells exposed to m-Tyr in vitro showed reduced cell viability, downregulated NFκB/STAT3/Notch axis, and induced autophagy; effects reversed by Phe. Strikingly, m-Tyr administration also impaired both, spontaneous metastasis derived from murine mammary carcinomas (4T1, C7HI, and LMM3) and PCa experimental metastases. Altogether, our findings propose m-Tyr delivery as a novel approach to boost the therapeutic efficacy of the current treatment for metastasis preventing the escape from tumor dormancy.

Resistencia concomitante antitumoral: un posible mecanismo de control de las metástasis / 1 CONCOMITANT TUMOR RESISTANCE: A POSIBLE CONTROL MECHANISM OF THE GROWTH OF METASTASES

La Resistencia Concomitante Antitumoral (RC) es el fenómeno según el cual un individuo portador de un tumor inhibe o retarda el crecimiento de implantes tumorales secundarios. Este fenómeno ha sido descripto en animales y en seres humanos y puede ser inducido tanto por tumores inmunogénicos como no-inmunogénicos. El estudio de la RC puede darnos indicios sobre mecanismos de control de las metástasis desde que las metástasis son, de hecho, implantes secundarios naturales desarrollados espontáneamente durante el crecimiento de un tumor primario. En este sentido la experiencia clínica y numerosos datos experimentales han revelado que la extirpación quirúrgica de un tumor puede ser seguida por una abrupta aceleración del crecimiento metastásico sugiriendo que, bajo ciertas circunstancias, un tumor puede ejercer un control inhibitorio sobre sus propias metástasis. En nuestro laboratorio hemos estudiado la RC asociada al crecimiento de numerosos tumores de ratón de diferente origen, tipo histológico e inmunogenicidad. Nuestros resultados demostraron que durante el crecimiento de un tumor primario se generan dos eventos temporalmente separados de RC. El primer evento es producido sólo por tumores inmunogénicos de pequeño tamaño (<500 mm3 ), es específico de tumor y es producido por mecanismos inmunológicos dependientes del timo. Por otro lado, el segundo evento de RC es inducido tanto por tumores inmunogénicos como no-inmunogénicos de gran tamaño (≥ 2000 mm3 ), no es específico de tumor, es timo independiente y correlaciona con la presencia de un factor sérico de bajo peso molecular que demostró tener capacidad para inhibir la proliferación de células tumorales tanto in vitro como in vivo. Cuando esta actividad antitumoral no estaba presente en el suero ­en nuestros modelos, los dos únicos casos estuvieron asociados a tumores altamente metastásicos­ el segundo evento de RC no se producía. Estos resultados sugieren una correlación directa entre la actividad sérica antitumoral, el segundo evento de RC y la capacidad para restringir el crecimiento metastásico.Aunque el primer evento de RC es producido, como dijimos arriba, por una respuesta inmunológica convencional mediada por células T, la naturaleza química del factor sérico asociado a la más universal manifestación de la RC (esto es, el segundo evento de RC), permaneció siendo un enigma por muchos años. En un trabajo reciente, identificamos ese factor sérico antitumoral como una mezcla de metatirosina y orto-tirosina, dos isómeros de tirosina que no están presentes en proteínas normales. Ambos isómeros fueron capaces de inhibir el crecimiento de diferentes tumores murinos que generan RC y restringieron el crecimiento de metástasis establecidas producidas por tumores que no generan RC pero son sensibles a la RC generada por otros tumores. A su vez, y tan significativo como lo anterior, estos efectos antitumorales se lograron sin ningún efecto colateral indeseado Una comprensión más profunda de los mecanismos moleculares asociados con el efecto antitumoral de estos isómeros de tirosina podría, eventualmente, ayudar a desarrollar métodos nuevos y menos tóxicos para combatir las enfermedades malignas; en particular para limitar el crecimiento acelerado de las metástasis después de la extirpación quirúrgica del tumor primario o después del padecimiento de traumas o estresores que pudieran ­despertar­ metástasis de su estado de ­tumor dormido­.

Concomitant tumor resistance (CR) is a phenomenon in which a tumor-bearing host is resistant to the growth of secondary tumor implants. This phenomenon has been described in human and animal systems and it can be generated by both immunogenic and non-immunogenic tumors. The relevance of CR to the mechanisms of metastases control has been highlighted by numerous observations showing that the removal of human and murine tumors may be followed by an abrupt increase in metastatic growth, suggesting that, upon certain circumstances, a primary tumor exerts a controlling action on its metastases which could be considered as secondary tumor implants developed spontaneously during the primary tumor growth. In our laboratory we have studied the CR induced by many murine tumors widely different in origin, histologic type and immunogenicity. Our results support the idea that during the primary tumor growth, there exist two temporally separate events of CR: the first one was exhibited only by small (<500 mm3 ) immunogenic tumors, it was tumor-specific and mediated by classical T-dependent immunological mechanisms. The second event was induced by both im munogenic and nonimmunogenic large (≥ 2000 mm3 ) tumors; it was non-tumor specific, thymus independent and correlated with the presence of a serum factor of low molecular weight that inhibited the in vitro and in vivo proliferation of tumor cells. When this anti-tumor serum activity was absent ­ in our hands, the only two cases were associated with highly metastatic tumors ­ the second event of CR did not exist, suggesting a direct correlation between the anti-tumor serum activity, the second event of CR and the ability to restrain metastatic growth. Although the mechanism associated with the first event of CR has, as said above, been elucidated as T cell­dependent, the molecular nature of the antitumor serum factor(s), which is at the root of the most universal manifestation of CR (that is, the second event of CR), remained an enigma for many years. In a recently published paper, we identified that antitumor serum factor(s) as a mixture of meta-tyrosine and ortho-tyrosine, two isomers of tyrosine that are not present in normal proteins. Both meta- and ortho-tyrosine inhibited the growth of different murine models of cancer that generate CR and could also block established spontaneous metastases produced by other murine models that do not generate CR but are very sensitive to the CR induced by other tumors. In addition, and most importantly, these anti-tumor effects were achieved without any collateral damage to the organism. A more profound understanding of the molecular mechanisms associated with the anti-tumor effects mediated by meta- and ortho-tyrosine could contribute to develop new and more harmless means to manage malignant diseases, especially by limiting the development of metastases that arise after resection of primary tumors or after other stressors that may promote the escape of metastases from dormancy.

B cells inhibit the antitumor immunity against an established murine fibrosarcoma.

Despite the classic role of B cells in favoring the immune response, an inhibitory action of B lymphocytes in tumor immunity has emerged in certain studies. In methylcolanthrene-induced murine fibrosarcoma (MCC), the loss of immunogenicity and the establishment of tolerance are paralleled by systemic immune suppression and the appearance of B+IL-10+ cells in tumor-draining lymph nodes. The present study aimed to assess the role of the B+IL-10+ cell population in the immune evasion and tolerance induced by MCC through the depletion of B cells in mice at various times of tumor progression: Prior to or subsequent to tumor implantation. Tumor growth and immunological parameters were evaluated. B cell depletion prior to tumor inoculum enhanced tumor growth, initiating the onset of the tumor-induced systemic immune response; however, an increase in the T regulatory cells (Tregs) at the tumor-draining lymph node could account for tumor exacerbation. B cell depletion once the tumor was established resulted in decreased tumor growth and a delayed onset of tolerance. Additionally, B cell absence exacerbated T cell dependent-tumor rejection, reduced Tregs and increased cytotoxic CD8+ T cells. In vitro analysis showed a direct effect of B cells upon T cell proliferation. In conclusion, B cell depletion exerts opposite effects when performed prior to or subsequent to tumor implantation. In this initially immunogenic tumor, B cell absence would delay the establishment of immunological tolerance probably by unmasking a pre-existing antitumor response. The present findings elucidate the convenience of modulating B cells in the development of future and more effective immunotherapies against cancer.

Methadone as first-line opioid treatment for cancer pain in a developing country palliative care unit.

PURPOSE: The use of methadone for cancer pain is limited by the need of expertise and close titration due to variable half-life. Yet, it is a helpful palliative strategy in low-resources countries given its long-acting effect at low cost and worth additional study. Our aim was to describe the prescription and outcomes of methadone as a first-line treatment for cancer pain in a tertiary palliative care unit (PCU) in Argentina. METHODS: Retrospective review of medical records of patients with moderate to severe cancer pain seen at the PCU in 1-year period, who initiated strong opioids at the first consultation. Data collected during the first month of treatment included disease and pain characteristics, initial and final opioid type and dose and need for opioid rotation. RESULTS: Methadone was the most frequent opioid both at the initial and last assessment (71 and 66 % of the prescriptions). In all, treatment with strong opioids provided considerable decrease in pain intensity (p < 0.001) with low and stable opioid dose. Median and interquartile range (IR) of oral morphine equivalent daily dose (OMEDD) was 26 (16-32) and 39 (32-55) mg for initial and final assessments, respectively (p = 0.3). In patients initiated with methadone, the median (IR) daily methadone dose was 5 (4-6) mg at first and 7.5 (6-10) mg at final assessment, and the median (IR) index of opioid escalation was 0 (0-4) mg; (p < 0.05). Patients on methadone underwent less percentage of opioid rotation (15 versus 50 %; p < 0.001) and longer time to rotation (20.6 ± 4.4 versus 9.0 ± 2.7 days; p < 0.001) than patients on other opioids. CONCLUSIONS: Results indicate the preference of methadone as first-line strong opioid treatment in a PCU, providing good pain relief at low doses with low need for rotation. Several considerations about the costs of strong opioids in the region are given.

Meta-tyrosine. A powerful anti-metastatic factor with undetectable toxic-side effects.

Concomitant tumor resistance (CR) is a phenomenon in which a tumor-bearing host is resistant to the growth of secondary tumor implants and metastasis. While former studies have indicated that T-cell dependent processes mediate CR in hosts bearing immunogenic small tumors, the most universal manifestation of CR induced by immunogenic and non-immunogenic large tumors had been associated with an antitumor serum factor that remained an enigma for many years. In a recent paper, we identified that elusive factor(s) as an equi-molar mixture of meta-tyrosine and ortho-tyrosine, two isomers of tyrosine that are not present in normal proteins and that proved to be responsible for 90% and 10%, respectively, of the total serum anti-tumor activity. In this work, we have extended our previous findings demonstrating that a periodic intravenous administration of meta-tyrosine induced a dramatic reduction of lung and hepatic metastases generated in mice bearing two different metastatic murine tumors and decreased the rate of death from 100% up to 25% in tumor-excised mice that already exhibited established metastases at the time of surgery. These anti-metastatic effects were achieved even at very low concentrations and without displaying any detectable toxic-side effects, suggesting that the use of meta-tyrosine may help to develop new and less harmful means of managing malignant diseases, especially those aimed to control the growth of metastases that is the most serious problem in cancer pathology.

Meta-tyrosine: A powerful anti-metastatic factor with undetectable toxic-side effects

Concomitant tumor resistance (CR) is a phenomenon in which a tumor-bearing host is resistant to the growth of secondary tumor implants and metastasis. While former studies have indicated that T-cell dependent processes mediate CR in hosts bearing immunogenic small tumors, the most universal manifestation of CR induced by immunogenic and non-immunogenic large tumors had been associated with an antitumor serum factor that remained an enigma for many years. In a recent paper, we identified that elusive factor(s) as an equi-molar mixture of meta-tyrosine and ortho-tyrosine, two isomers of tyrosine that are not present in normal proteins and that proved to be responsible for 90% and 10%, respectively, of the total serum anti-tumor activity. In this work, we have extended our previous findings demonstrating that a periodic intravenous administration of meta-tyrosine induced a dramatic reduction of lung and hepatic metastases generated in mice bearing two different metastatic murine tumors and decreased the rate of death from 100% up to 25% in tumor-excised mice that already exhibited established metastases at the time of surgery. These anti-metastatic effects were achieved even at very low concentrations and without displaying any detectable toxic-side effects, suggesting that the use of meta-tyrosine may help to develop new and less harmful means of managing malignant diseases, especially those aimed to control the growth of metastases that is the most serious problem in cancer pathology.

La resistencia concomitante antitumoral (RC) es el fenómeno según el cual un individuo portador de tumor inhibe el crecimiento de implantes tumorales secundarios y metástasis. Si bien desde hace tiempo se sabe que la RC inducida por tumores inmunogénicos de pequeño tamaño es generada por mecanismos inmunológicos dependientes de células T, por otro lado, la manifestación más universal de la RC, generada tanto por tumores inmunogénicos como no-inmunogénicos de gran tamaño, había sido asociada con un (unos) factor sérico antitumoral cuya naturaleza permaneció elusiva por años. En un trabajo reciente, nuestro grupo de trabajo identificó este factor como la mezcla equi-molar de meta-tirosina y orto-tirosina, dos isómeros de tirosina que no están presentes en proteínas normales y que demostraron ser responsables del 90% y 10%, respectivamente, de la actividad antitumoral total del suero. En este trabajo, continuamos nuestras investigaciones demostrando que la administración periódica de meta-tirosina reducía drásticamente el número de metástasis pulmonares y hepáticas en ratones portadores de dos tumores murinos altamente metastásicos y disminuía dramáticamente la mortandad (de 100% a 25%) de ratones con metástasis ya establecidas al momento de la extirpación quirúrgica del tumor. Estos efectos anti-metastásicos se lograron aun con muy bajas concentraciones de meta-tirosina y sin efectos tóxicos perceptibles, lo que sugiere que su uso puede ayudar a diseñar nuevas y menos nocivas estrategias para el tratamiento del cáncer, especialmente aquellas destinadas a controlar el crecimiento metastásico, que es el problema más grave en la enfermedad oncológica.

Meta-tyrosine: A powerful anti-metastatic factor with undetectable toxic-side effects

Concomitant tumor resistance (CR) is a phenomenon in which a tumor-bearing host is resistant to the growth of secondary tumor implants and metastasis. While former studies have indicated that T-cell dependent processes mediate CR in hosts bearing immunogenic small tumors, the most universal manifestation of CR induced by immunogenic and non-immunogenic large tumors had been associated with an antitumor serum factor that remained an enigma for many years. In a recent paper, we identified that elusive factor(s) as an equi-molar mixture of meta-tyrosine and ortho-tyrosine, two isomers of tyrosine that are not present in normal proteins and that proved to be responsible for 90% and 10%, respectively, of the total serum anti-tumor activity. In this work, we have extended our previous findings demonstrating that a periodic intravenous administration of meta-tyrosine induced a dramatic reduction of lung and hepatic metastases generated in mice bearing two different metastatic murine tumors and decreased the rate of death from 100% up to 25% in tumor-excised mice that already exhibited established metastases at the time of surgery. These anti-metastatic effects were achieved even at very low concentrations and without displaying any detectable toxic-side effects, suggesting that the use of meta-tyrosine may help to develop new and less harmful means of managing malignant diseases, especially those aimed to control the growth of metastases that is the most serious problem in cancer pathology.(AU)

La resistencia concomitante antitumoral (RC) es el fenómeno según el cual un individuo portador de tumor inhibe el crecimiento de implantes tumorales secundarios y metástasis. Si bien desde hace tiempo se sabe que la RC inducida por tumores inmunogénicos de pequeño tamaño es generada por mecanismos inmunológicos dependientes de células T, por otro lado, la manifestación más universal de la RC, generada tanto por tumores inmunogénicos como no-inmunogénicos de gran tamaño, había sido asociada con un (unos) factor sérico antitumoral cuya naturaleza permaneció elusiva por años. En un trabajo reciente, nuestro grupo de trabajo identificó este factor como la mezcla equi-molar de meta-tirosina y orto-tirosina, dos isómeros de tirosina que no están presentes en proteínas normales y que demostraron ser responsables del 90% y 10%, respectivamente, de la actividad antitumoral total del suero. En este trabajo, continuamos nuestras investigaciones demostrando que la administración periódica de meta-tirosina reducía drásticamente el número de metástasis pulmonares y hepáticas en ratones portadores de dos tumores murinos altamente metastásicos y disminuía dramáticamente la mortandad (de 100% a 25%) de ratones con metástasis ya establecidas al momento de la extirpación quirúrgica del tumor. Estos efectos anti-metastásicos se lograron aun con muy bajas concentraciones de meta-tirosina y sin efectos tóxicos perceptibles, lo que sugiere que su uso puede ayudar a diseñar nuevas y menos nocivas estrategias para el tratamiento del cáncer, especialmente aquellas destinadas a controlar el crecimiento metastásico, que es el problema más grave en la enfermedad oncológica.(AU)

Regulatory B cells present in lymph nodes draining a murine tumor.

In cancer, B cells have been classically associated with antibody secretion, antigen presentation and T cell activation. However, a possible role for B lymphocytes in impairing antitumor response and collaborating with tumor growth has been brought into focus. Recent reports have described the capacity of B cells to negatively affect immune responses in autoimmune diseases. The highly immunogenic mouse tumor MCC loses its immunogenicity and induces systemic immune suppression and tolerance as it grows. We have previously demonstrated that MCC growth induces a distinct and progressive increase in B cell number and proportion in the tumor draining lymph nodes (TDLN), as well as a less prominent increase in T regulatory cells. The aim of this research was to study B cell characteristics and function in the lymph node draining MCC tumor and to analyze whether these cells may be playing a role in suppressing antitumor response and favoring tumor progression. Results indicate that B cells from TDLN expressed increased CD86 and MHCII co-stimulatory molecules indicating activated phenotype, as well as intracellular IL-10, FASL and Granzyme B, molecules with regulatory immunosuppressive properties. Additionally, B cells showed high inhibitory upon T cell proliferation ex vivo, and a mild capacity to secrete antibodies. Our conclusion is that even when evidence of B cell-mediated activity of the immune response is present, B cells from TDLN exhibit regulatory phenotype and inhibitory activity, probably contributing to the state of immunological tolerance characteristic of the advanced tumor condition.

Regulatory B cells present in lymph nodes draining a murine tumor

In cancer, B cells have been classically associated with antibody secretion, antigen presentation and T cell activation. However, a possible role for B lymphocytes in impairing antitumor response and collaborating with tumor growth has been brought into focus. Recent reports have described the capacity of B cells to negatively affect immune responses in autoimmune diseases. The highly immunogenic mouse tumor MCC loses its immunogenicity and induces systemic immune suppression and tolerance as it grows. We have previously demonstrated that MCC growth induces a distinct and progressive increase in B cell number and proportion in the tumor draining lymph nodes (TDLN), as well as a less prominent increase in T regulatory cells. The aim of this research was to study B cell characteristics and function in the lymph node draining MCC tumor and to analyze whether these cells may be playing a role in suppressing antitumor response and favoring tumor progression. Results indicate that B cells from TDLN expressed increased CD86 and MHCII co-stimulatory molecules indicating activated phenotype, as well as intracellular IL-10, FASL and Granzyme B, molecules with regulatory immunosuppressive properties. Additionally, B cells showed high inhibitory upon T cell proliferation ex vivo, and a mild capacity to secrete antibodies. Our conclusion is that even when evidence of B cell-mediated activity of the immune response is present, B cells from TDLN exhibit regulatory phenotype and inhibitory activity, probably contributing to the state of immunological tolerance characteristic of the advanced tumor condition.

Regulatory B cells present in lymph nodes draining a murine tumor. / Regulatory B cells present in lymph nodes draining a murine tumor.

In cancer, B cells have been classically associated with antibody secretion, antigen presentation and T cell activation. However, a possible role for B lymphocytes in impairing antitumor response and collaborating with tumor growth has been brought into focus. Recent reports have described the capacity of B cells to negatively affect immune responses in autoimmune diseases. The highly immunogenic mouse tumor MCC loses its immunogenicity and induces systemic immune suppression and tolerance as it grows. We have previously demonstrated that MCC growth induces a distinct and progressive increase in B cell number and proportion in the tumor draining lymph nodes (TDLN), as well as a less prominent increase in T regulatory cells. The aim of this research was to study B cell characteristics and function in the lymph node draining MCC tumor and to analyze whether these cells may be playing a role in suppressing antitumor response and favoring tumor progression. Results indicate that B cells from TDLN expressed increased CD86 and MHCII co-stimulatory molecules indicating activated phenotype, as well as intracellular IL-10, FASL and Granzyme B, molecules with regulatory immunosuppressive properties. Additionally, B cells showed high inhibitory upon T cell proliferation ex vivo, and a mild capacity to secrete antibodies. Our conclusion is that even when evidence of B cell-mediated activity of the immune response is present, B cells from TDLN exhibit regulatory phenotype and inhibitory activity, probably contributing to the state of immunological tolerance characteristic of the advanced tumor condition.

Concomitant tumor resistance.

Concomitant tumor resistance (CR) is a phenomenon in which a tumor-bearing host is resistant to the growth of secondary tumor implants. This phenomenon has been described in human and animal systems and it can be generated by both immunogenic and non-immunogenic tumors. The relevance of CR to the mechanisms of metastases control has been highlighted by numerous observations showing that the removal of human and murine tumors may be followed by an abrupt increase in metastatic growth, suggesting that a primary tumor may exert a controlling action on its metastases which could be considered as secondary tumor implants developed spontaneously during the primary tumor growth. A more profound understanding of the different mechanisms claimed to be associated with the phenomenon of CR could contribute to develop new and more harmless means to manage malignant diseases, especially by limiting the development of metastases that arise after resection of primary tumors or after other stressors that may promote the escape of metastases from dormancy.

Concomitant tumor resistance: the role of tyrosine isomers in the mechanisms of metastases control.

Concomitant tumor resistance (CR) is a phenomenon in which a tumor-bearing host is resistant to the growth of secondary tumor implants and metastasis. Although previous studies indicated that T-cell-dependent processes mediate CR in hosts bearing immunogenic small tumors, manifestations of CR induced by immunogenic and nonimmunogenic large tumors have been associated with an elusive serum factor. In a recently published study, we identified this factor as meta-tyrosine and ortho-tyrosine, 2 isomers of tyrosine that would not be present in normal proteins. In 3 different murine models of cancer that generate CR, both meta- and ortho-tyrosine inhibited tumor growth. Additionally, we showed that both isoforms of tyrosine blocked metastasis in a fourth model that does not generate CR but is sensitive to CR induced by other tumors. Mechanistic studies showed that the antitumor effects of the tyrosine isomers were mediated in part by early inhibition of the MAP/ERK pathway and inactivation of STAT3, potentially driving tumor cells into a state of dormancy in G(0)-phase. Other mechanisms, putatively involving the activation of an intra-S-phase checkpoint, would also inhibit tumor proliferation by accumulating cells in S-phase. By revealing a molecular basis for the classical phenomenon of CR, our findings may stimulate new generalized approaches to limit the development of metastases that arise after resection of primary tumors or after other stressors that may promote the escape of metastases from dormancy, an issue that is of pivotal importance to oncologists and their patients.

On the immunostimulatory hypothesis of cancer.

There is a rather generalized belief that the worst possible outcome for the application of immunological therapies against cancer is a null effect on tumor growth. However, a significant body of evidence summarized in the immunostimulatory hypothesis of cancer suggests that, upon certain circumstances, the growth of incipient and established tumors can be accelerated rather than inhibited by the immune response supposedly mounted to limit tumor growth. In order to provide more compelling evidence of this proposition, we have explored the growth behavior characteristics of twelve murine tumors -most of them of spontaneous origin- arisen in the colony of our laboratory, in putatively immunized and control mice. Using classical immunization procedures, 8 out of 12 tumors were actually stimulated in "immunized" mice while the remaining 4 were neither inhibited nor stimulated. Further, even these apparently non-antigenic tumors could reveal some antigenicity if more stringent than classical immunization procedures were used. This possibility was suggested by the results obtained with one of these four apparently non-antigenic tumors: the LB lymphoma. In effect, upon these stringent immunization pretreatments, LB was slightly inhibited or stimulated, depending on the titer of the immune reaction mounted against the tumor, with higher titers rendering inhibition and lower titers rendering tumor stimulation. All the above results are consistent with the immunostimulatory hypothesis that entails the important therapeutic implications -contrary to the orthodoxy- that, anti-tumor vaccines may run a real risk of doing harm if the vaccine-induced immunity is too weak to move the reaction into the inhibitory part of the immune response curve and that, a slight and prolonged immunodepression -rather than an immunostimulation- might interfere with the progression of some tumors and thus be an aid to cytotoxic therapies.

Tyrosine isomers mediate the classical phenomenon of concomitant tumor resistance.

Concomitant tumor resistance (CR) is a phenomenon originally described in 1906 in which a tumor-bearing host is resistant to the growth of secondary tumor implants and metastasis. Although recent studies have indicated that T-cell-dependent processes mediate CR in hosts bearing immunogenic small tumors, manifestations of CR induced by immunogenic and nonimmunogenic large tumors have been associated with an elusive serum factor. In this study, we identify this serum factor as tyrosine in its meta and ortho isoforms. In three different murine models of cancer that generate CR, both meta-tyrosine and ortho-tyrosine inhibited tumor growth. In addition, we showed that both isoforms of tyrosine blocked metastasis in a fourth model that does not generate CR but is sensitive to CR induced by other tumors. Mechanistic studies showed that the antitumor effects of the tyrosine isoforms were mediated, in part, by early inhibition of mitogen-activated protein/extracellular signal-regulated kinase pathway and inactivation of STAT3, potentially driving tumor cells into a state of dormancy. By revealing a molecular basis for the classical phenomenon of CR, our findings may stimulate new generalized approaches to limit the development of metastases that arise after resection of primary tumors, an issue of pivotal importance to oncologists and their patients.

Lymphadenectomy exacerbates tumor growth while lymphadenectomy plus the adoptive transfer of autologous cytotoxic cells and low-dose cyclophosphamide induces regression of an established murine fibrosarcoma.

Tumor-draining lymph node (TDLN) ablation is routinely performed in the management of cancer; nevertheless, its usefulness is at present a matter of debate. TDLN are central sites where T cell priming to tumor antigens and onset of the antitumor immune response occur. However, tumor-induced immunosuppression has been demonstrated at TDLN, leading to downregulation of antitumor reaction and tolerance induction. Tolerance in turn is a main impairment for immunotherapy trials. We used a murine immunogenic fibrosarcoma that evolves to a tolerogenic state, to study the cellular and molecular mechanisms underlying tolerance induction at the level of TDLN and to design an appropriate immunotherapy. We determined that following a transient activation, the established tumor induces signs of immunosuppression at TDLN that coexist with local and systemic evidences of antitumor response. Therefore, we evaluated the feasibility of removing TDLN in order to eliminate a focus of immunosuppression and favor tumor rejection; but instead, a marked exacerbation of tumor growth was induced. Combining TDLN ablation with the in vivo depletion of regulatory cells by low-dose cyclophosphamide and the restoring of the TDLN-derived cells into the donor mouse by adoptive transference, resulted in lowered tumor growth, enhanced survival and a considerable degree of tumor regression. Our results demonstrate that important antitumor elements can be eliminated by lymphadenectomy and proved that the concurrent administration of low-dose chemotherapy along with the reinoculation of autologous cytotoxic cells provides protection. We suggest that this protocol may be useful, especially in the cases where lymphadenectomy is mandatory.

Biphasic effect of a primary tumor on the growth of secondary tumor implants.

BACKGROUND: The phenomenon of hormesis is characterized by a biphasic dose-response, exhibiting opposite effects in the low- and high-dose zones. In this study, we explored the possibility that the hormesis concept may describe the interactions between two tumors implanted in a single mouse, such that the resulting tumors are of different sizes. MATERIALS AND METHODS: We used two murine tumors of spontaneous origin and undetectable immunogenicity growing in BALB/c mice. A measure of cell proliferation was obtained by immunostaining for Ki-67 protein and by using the [(3)H] thymidine uptake assay. For serum fractionation, we utilized dialysis and chromatography on Sephadex G-15. RESULTS: The larger primary tumor induced inhibitory or stimulatory effects on the growth of the smaller secondary one, depending on the ratio between the mass of the larger tumor relative to that of the smaller one, with high ratios rendering inhibition and low ratios inducing stimulation of the secondary tumor. CONCLUSION: Since metastases can be considered as natural secondary tumor implants in a tumor-bearing host and that they constitute the main problem in cancer pathology, the use of the concept of hormesis to describe those biphasic effects might have significant clinical implications. In effect, if the tumor-bearing host were placed in the inhibitory window, tumor extirpation could enhance the growth of distant metastases and, reciprocally, if placed in the stimulatory window, tumor extirpation would result not only in a reduction or elimination of primary tumor load but also in a slower growth or inhibition of metastases.