Bortezomib-thalidomide-dexamethasone-cisplatin-doxorubicin-cyclophosphamide-etoposide as a Salvage and Bridging Regimen before Hematopoietic Stem Cell Transplantation for Relapsed or Refractory Multiple Myeloma.

Objective Currently, treatment of relapsed or refractory multiple myeloma is challenging. Although bortezomib-thalidomide-dexamethasone-cisplatin-doxorubicin-cyclophosphamide-etoposide (VTD-PACE), a potent combination of a proteasome inhibitor, immunomodulatory drug, and conventional chemotherapeutics, is a widely used regimen, its efficacy and safety are unclear. Methods We retrospectively analyzed the clinical data of 35 patients treated with VTD-PACE. Results The overall response rate was 65.7% (complete response, 5.7%). The median progression-free survival (PFS) and overall survival (OS) were 8.0 [95% confidence interval (CI), 0.9-15.0] and 20.0 (95% CI, 17.5-22.5) months, respectively. Twenty-two (62.9%) patients developed grade 3-4 infections, and no therapy-related deaths occurred. Sixteen of 25 patients (64%) underwent stem cell harvest successfully with more than 2.0×106/kg of CD34 cells after VTD-PACE. Twenty-two patients underwent autologous or allogeneic stem cell transplantation (SCT). The response and survival durations were short in patients without SCT after VTD-PACE [median PFS: 4.0 (95% CI, 2.7-5.3) months; OS: 14.0 (6.9-21.0) months]; however, these responses significantly improved with SCT following VTD-PACE. The PFS was 8.0 (NA) months (p=0.024), and the OS was 21.0 (19.1-22.8) months (p=0.019). Conclusion VTD-PACE is an effective and tolerable salvage regimen and feasible bridging therapy for SCT.

Achievements and challenges of the Sakigake designation system in Japan.

The Sakigake designation system (Sakigake) has been launched to encourage the pioneered development of innovative new medical products for the effective treatment of severe illness in Japan, which allows leveraging the several advantages in prioritized consultation, rapid review, premium drug pricing and extended data-protection period. We retrospectively analysed the Sakigake products including drugs and regenerative medical products to clarify the achievements and the future issues in this system. From April 2015 to August 2020 (the first 5-year trial period of Sakigake), 37 products were designated, and 10 of those were approved in Japan in which 7 new active substances achieved the first-in-world approvals. Oncology, neurology and cardiovascular disease were the major therapeutic areas, and those 3 accounted for 75.7% of all products. Sakigake achieved some first-in-world approvals by the Pharmaceuticals and Medical Devices Agency/the Ministry of Health, Labor and Welfare of innovative new medical products, although in some therapeutic areas, there remains room in stimulating drug development.

Survival case of rhinocerebral and pulmonary mucormycosis due to Cunninghamella bertholletiae during chemotherapy for acute myeloid leukemia: a case report.

A 42-year-old man diagnosed with acute myeloid leukemia complained of progressive swelling of the right side of his face with pain 11 days after the third cycle of consolidation therapy with high-dose arabinosylcytosine-cytarabine. Head and neck magnetic resonance imaging showed a mass lesion in his right maxillary sinus with parapharyngeal involvement, which included the right masseter muscle, intraorbital involvement, and an abscess in his brain. Chest computed tomography revealed peribronchial small nodules in his right upper lobe and a necrotic tumor in his right lower lobe. Molds identified as Cunninghamella bertholletiae were isolated from the necrotic ulcer. According to these results, chemotherapy for leukemia was discontinued. High-dose liposomal amphotericin (10 mg/kg/day) was initiated. Because renal dysfunction occurred, the dosage was decreased to 6 mg/kg and combined with 150 mg/day micafungin. Debridement of necrotic tissue in the right maxillary sinus and establishment of the fenestration between the sinus and oral cavity were performed. Subsequently, brain and lung lesions were surgically removed. Rhinocerebral mucormycosis was successfully treated without relapse over 3 years by a 112-day course of intravenous anti-fungal therapy and 223-day course of terbinafine and partial surgical removal, respectively, to maintain masticatory and ocular functions. To our knowledge, there has been no other report of a long-term survival case of rhinocerebral mucormycosis due to C. bertholletiae.

Evolving Landscape of New Drug Approval in Japan and Lags from International Birth Dates: Retrospective Regulatory Analysis.

The Pharmaceuticals and Medical Devices Agency (PMDA) has approved hundreds of new drugs in recent years. We retrospectively analyzed the new drugs approved in Japan from 2008 to 2019, and identify the first-in-world approvals and clarify the current drug lag. The new drug and the drug lag were defined as a drug with a new active substance and a difference between the approval date in Japan and the international birth date, respectively. Among 400 new drugs approved in Japan during the last 12 years, 80 (20.0%) were first approved in Japan, and 320 were outside Japan (the United States: 202, 50.5%; Europe: 82, 20.5%; other regions: 36, 9.0%). Of these, 45 new drugs have not yet been approved outside Japan, and the remaining 355 have been globally approved in Japan and overseas. The number of new drug approvals were the largest in oncology followed by metabolic/endocrine and infectious diseases. The median drug lags (year) among all 400 new drugs and 355 new drugs with global approvals were 4.3 and 4.7 in the first tertile (2008-2011), 1.5 and 2.6 in the second tertile (2012-2015), and reduced to 1.3 and 2.2 in the third tertile (2016-2019), respectively. Substantial drug lag remains in neurology, psychiatry, and therapeutic areas where the number of new drug approvals was relatively small. Collectively, one-fifth of the new drugs approved in Japan are first-in-world approvals. Drug lag has been greatly decreased, although it still exists.

Efficacy of VRD(Bortezomib, Lenalidomide, and Dexamethasone) Consolidation Therapy and Maintenance Therapy with Immunomodulatory Drugs(Thalidomide or Lenalidomide) after Autologous Peripheral Blood Stem Cell Transplantation in the Era of Bortezomib-Containing Induction Therapy-A Single Institution Experience.

Autologous stem cell transplantation(ASCT)for newly-diagnosed multiple myeloma(NDMM)has underwent recent improvements in combination with novel agents-containing induction and post-ASCT therapy. Since the approval of bortezomib for NDMM in Japan, we conducted the following regimen(BD arm)in transplant-eligible patients with NDMM: BD (bortezomib and dexamethasone)induction, ASCT, VRD consolidation, and maintenance therapy with immunomodulatory drugs(IMIDs). The efficacy and safety of the BD arm were compared to those of patients treated with vincristine, doxorubicin, and dexamethasone(VAD)induction followed by ASCT(VAD arm)retrospectively. Thirty-three patients were treated with the BD arm, and 92 patients with the VAD arm. Thirty-one patients in the BD arm proceeded to ASCT. Thereafter, 23 and 17 patients received VRD consolidation and IMIDs maintenance therapy, respectively. The rates of complete response/Bvery good partial response after ASCT, consolidation, and maintenance therapy were 43%/61%, 76%/90% and 87%/93%, respectively. The response rates after ASCT did not differ between BD and VAD arms. The median PFS was 46.2 months(BD arm)and 30.6 months(VAD arm)(HR 0.48[0.27-0.85], p=0.0106). The median OS was not-reached(BD arm)and 90.6 months(VAD arm)(HR 0.21[0.05-0.87], p=0.0172). VRD consolidation and IMIDs maintenance therapies improved disease status after ASCT and prolonged PFS and OS.

Genetic aberrations in iPSCs are introduced by a transient G1/S cell cycle checkpoint deficiency.

A number of point mutations have been identified in reprogrammed pluripotent stem cells such as iPSCs and ntESCs. The molecular basis for these mutations has remained elusive however, which is a considerable impediment to their potential medical application. Here we report a specific stage at which iPSC generation is not reduced in response to ionizing radiation, i.e. radio-resistance. Quite intriguingly, a G1/S cell cycle checkpoint deficiency occurs in a transient fashion at the initial stage of the genome reprogramming process. These cancer-like phenomena, i.e. a cell cycle checkpoint deficiency resulting in the accumulation of point mutations, suggest a common developmental pathway between iPSC generation and tumorigenesis. This notion is supported by the identification of specific cancer mutational signatures in these cells. We describe efficient generation of human integration-free iPSCs using erythroblast cells, which have only a small number of point mutations and INDELs, none of which are in coding regions.

Color-coded Imaging of the Fate of Cancer-cell-derived Exosomes During Pancreatic Cancer Metastases in a Nude-mouse Model.

BACKGROUND/AIM: Tumor-derived exosomes play important roles in tumor metastases. In this report, we observed the fate of tumor-derived exosomes in pancreatic cancer metastatic nude-mouse models using color-coded imaging. MATERIALS AND METHODS: Mia-PaCa-2 human pancreatic cancer cells expressing red fluorescent protein (RFP) were transduced by exosome-specific pCT-CD63-green fluorescent protein (GFP) and injected in the spleen of nude mice. RESULTS: Four weeks after injection of these cells into the spleen, liver metastases developed and tumor-derived exosomes were observed within the metastatic cancer cells and in Kupffer cells. Furthermore, tumor-derived exosomes diffused to bone marrow and lung cells, especially macrophages, without any metastases present. CONCLUSION: In the present study, we visualized the distribution of cancer-derived exosomes for the first time at the cellular level, in a pancreatic-cancer metastatic model.

Choline-deficient-diet Decreases Fibroblasts in the Circulating Tumor Cell (CTC) Microenvironment.

BACKGROUND/AIM: Circulating tumor cells (CTCs) may have an important role in metastasis. CTC clusters, which contain fibroblasts, indicate poor prognosis. In the present study, we used our malignant lymphoma metastatic mouse model to compare the effect of a choline-deficient-diet (CDD) and the control diet (CD) on fibroblasts in CTCs. MATERIALS AND METHODS: We compared the number and morphology of CTCs in CDD and CD mice using color-coded imaging with fluorescent proteins. Malignant lymphoma EL4 cells expressing RFP were injected in the spleen of transgenic C57B/6-GFP mice, which were fed a CDD or CD. Two weeks later, we harvested and observed the number of CTCs and fibroblast-like cells both in heart blood and portal blood. Imaging of CTC morphology was performed with smeared glass slides and in culture. RESULTS AND CONCLUSION: There was no significant difference in the number of CTCs between CDD and CD mice. The number of fibroblast-like cells in the CTC microenvironment in CD mouse portal blood was significantly larger than in CDD mouse portal blood. These differences may be caused by deficiency in choline that leads to less metastasis in choline-deficient-diet-induced fatty liver.

Color-coded Imaging of the Circulating Tumor Cell Microenvironment.

BACKGROUND/AIM: Circulating tumor cells (CTCs) may initiate metastasis. Some studies show that the number of CTCs and existence of CTC clusters can be prognostic. In the present study, our color-coded imaging nude mouse model of metastatic lymphoma was utilized to investigate the microenvironment of CTC clusters using fluorescent-protein imaging. MATERIALS AND METHODS: EL-4 mouse lymphoma cells expressing red fluorescent protein (RFP) were injected into the spleen of transgenic C57B/6-green fluorescent protein (GFP) mice. Three weeks later, the number of CTCs and CTC clusters both in heart blood and portal blood were quantified and characterized using confocal microscopy for color-coded imaging. RESULTS: There was no significant difference in the number of CTCs between heart and portal blood. CTC clusters comprised 8.8% of CTCs, determined by color-coded imaging. Heterotypic CTC clusters containing other types of cells were distinguishable from homotypic CTCs. Heterotypic CTC clusters comprising cancer cells and fibroblasts were more rare than homotypic ones. Heterotypic CTC clusters with fibroblasts were observed only in portal blood, not in heart blood. CONCLUSION: CTCs can have variable properties depending on the blood source. CTCs can form clusters, which may contain fibroblast that may play a role in promoting CTC metastasis. Our results demonstrate the concept of the CTC microenvironment (CME), which may play a critical role in CTC behavior, including of metastasis.

The Prognostic Impact of Dose-attenuated R-CHOP Therapy for Elderly Patients with Diffuse Large B-cell Lymphoma.

Objective Although R-CHOP (rituximab, cyclophosphamide, vincristine, doxorubicin, and prednisone) is a standard therapy for diffuse large B-cell lymphoma (DLBCL), the optimal dose for elderly patients remains unclear. Methods and Patients We retrospectively verified our R-CHOP dose-attenuation system implemented from 2005 for DLBCL patients. Among the 115 DLBCL patients treated during 2001-2010, 33 patients treated during 2001-2005 received R-CHOP doses adjusted according to physicians' decisions (PHY group). Eighty-two patients treated after 2005 received adjusted R-CHOP doses according to a unified dose-attenuation system (UNI group). Patients aged <60, 60-69, 70-79, and ≥80 years received the standard R-CHOP, 100% R-CHO+P (50 mg/m2), 100% R+75% CHO+P (40 mg/m2), and 100% R+50% CHO+P (30 mg/m2), respectively. We compared the responses, survival, and treatment cessation between the PHY and UNI groups. Results The patients' characteristics between both groups were closely comparable. All PHY patients received randomly adjusted R-CHOP doses; 94% of UNI patients received scheduled doses. The complete response rates differed significantly between the UNI (77%) and PHY patients (50%) (p=0.011). The two-year event-free survival rates were 50% and 32% in the UNI and PHY groups, respectively (p=0.0083). The two-year OS rates were 77% and 72% in the UNI and PHY group (p=0.16). Among the patients aged >70 years (n=59) overall survival was shorter in the PHY group (62%) than in the UNI group (72%; p=0.02). The UNI group received higher anti-tumor agent doses than the PHY group. The therapy discontinuation rates were 5% in the UNI group and 24% in the PHY group. Conclusion Carrying out unified dose reduction may improve the efficacy and prognosis among elderly DLBCL patients.

Color-coded Imaging Distinguishes Cancer Cells, Stromal Cells, and Recombinant Cancer-stromal Cells in the Tumor Microenvironment During Metastasis.

BACKGROUND/AIM: Our laboratory pioneered color-coded imaging of the tumor microenvironment (TME). We observed recruitment of cancer and stromal cells to the TME and recombination between cancer and stromal cells. The aim of the present study was to observe the dynamics of the TME by color-coded imaging during metastasis and in the formation of a pre-metastatic niche. MATERIALS AND METHODS: Red-fluorescent protein (RFP-expressing) mouse colon-cancer 26 cells were initially injected subcutaneously in green-fluorescent protein (GFP) nude mice. The resulting subcutaneous tumors were harvested and cultured. The cultured subcutaneous tumors contained RFP colon cancer cells, GFP stromal cells and recombinant cancer-stromal cells expressing yellow fluorescence. After 14 days culture, the cells were injected into the spleen. RESULTS: After splenic injection, colon-cancer 26 metastases were observed in the liver, ascites, and bone marrow. Using the Olympus FV1000 confocal microscope, the cells cultured from tumors and metastasis in each site were visualized. RFP colon-cancer cells, GFP stromal cells derived from host GFP nude mice, and recombinant yellow-fluorescent cells were observed in each organ. In addition, in the liver, areas with only GFP stromal cells were observed and assumed to be a pre-metastatic niche. CONCLUSION: Color-coded imaging demonstrated the dynamics of colon cancer and stromal cells at different metastatic sites including the formation of recombinant cancer-stromal cells.

Differential Organ-targeting and Cellular Characteristics of Metastatic Human Pancreatic Cancer Cell Lines in Mouse Models.

BACKGROUND/AIM: The lethal characteristic of pancreatic cancer is metastasis which is recalcitrant to currently-used chemotherapy. Our aim was to understand metastasis at the cellular level. We previously reported that multi-nucleate cells or spindle cells were more prominent in pancreatic cancer metastasis than in the primary tumor. In the present report, we investigated four representative human pancreatic cell lines for differences in cell morphology between the primary tumor and various metastatic organ targets for each cell line. MATERIALS AND METHODS: Human pancreatic cancer cell lines AsPC-1, Panc-1, KP2 and KP3 were used. Pancreatic cancer cells were injected into spleen of nude mice resulting in experimental metastasis to various organs which were observed at the cellular level when the organs were placed into culture. RESULTS: AsPC-1 and KP2 pancreatic cells formed many experimental liver metastases, in contrast to Panc-1 and KP3. Lung metastasis was only observed for AsPC-1. In the cultures established from the primary and metastatic tumors, multi-nucleate cells were found to be more prominent in the metastasis of the pancreatic cell lines with frequent metastasis, AsPC-1 and KP2. Spindle-like cells were observed prominently in AsPC-1 lung metastasis. CONCLUSION: Human pancreatic cancer cell lines have differential metastatic characteristics with regard to target organs and cell-morphology changes. Multi-nucleate and spindle cells may play an important role in pancreatic cancer metastasis to the liver and lung, respectively.

Improved progression-free and event-free survival in myeloma patients undergoing PBSCH receiving a cyclophosphamide + G-CSF regimen than G-CSF alone.

Two regimens are commonly used for peripheral blood hematopoietic stem cell harvesting (PBSCH) in multiple myeloma: high-dose cyclophosphamide (HD-CY) + granulocyte-colony stimulating factor (G-CSF), and G-CSF alone. The objective of the present study was to evaluate the anti-myeloma effect of the PBSCH regimen including HD-CY. We retrospectively assessed harvesting efficiency, complications, and anti-myeloma effects in 115 patients receiving HD-CY + G-CSF (HD-CY group) and 32 patients receiving G-CSF alone (G-alone group). We collected > 2 × 106 CD34-positive cells/kg from 93 and 75% of patients in the HD-CY and G-alone groups, respectively (P = 0.0079). The mean HSC count was also higher in the HD-CY group. No severe complications were observed in the G-alone group, whereas 66% of patients in the HD-CY group were treated with intravenous antibiotics. The median progression-free and event-free survival (PFS and EFS) were longer in the HD-CY group than in the G-alone group (28 vs. 18 months and 25 vs. 13 months, respectively; P = 0.0127 and 0.0139), with no difference in median overall survival. HD-CY showed anti-myeloma effect, as verified by prolonged EFS and PFS, when a vincristine, doxorubicin, and dexamethasone regimen was administered as induction before PBSCH.

Inhibition of CTGF ameliorates peritoneal fibrosis through suppression of fibroblast and myofibroblast accumulation and angiogenesis.

Peritoneal fibrosis (PF) is a serious complication in various clinical settings, but the mechanisms driving it remain to be fully determined. Connective tissue growth factor (CTGF) is known to regulate fibroblast activities. We therefore examined if CTGF inhibition has anti-fibrotic effects in PF. PF was induced by repetitive intraperitoneal injections of chlorhexidine gluconate (CG) in mice with type I pro-collagen promoter-driven green fluorescent protein (GFP) expression to identify fibroblasts. FG-3019, an anti-CTGF monoclonal antibody, was used to inhibit CTGF. CG-induced PF was significantly attenuated in FG-3019-treated mice. CG challenges induced marked accumulations of proliferating fibroblasts and of myofibroblasts, which were both reduced by FG-3019. Levels of peritoneal CTGF expression were increased by CG challenges, and suppressed in FG-3019-treated mice. FG-3019 treatment also reduced the number of CD31+ vessels and VEGF-A-positive cells in fibrotic peritoneum. In vitro studies using NIH 3T3 fibroblasts and peritoneal mesothelial cells (PMCs) showed that CTGF blockade suppressed TGF-ß1-induced fibroblast proliferation and myofibroblast differentiation, PMC mesothelial-to-mesenchymal transition, and VEGF-A production. These findings suggest that the inhibition of CTGF by FG-3019 might be a novel treatment for PF through the regulation of fibroblast and myofibroblast accumulation and angiogenesis.

Choline-Deficient-Diet-Induced Fatty Liver Is a Metastasis-Resistant Microenvironment.

BACKGROUND/AIM: Fatty liver disease is increasing in the developed and developing world. Liver metastasis from malignant lymphoma in the fatty liver is poorly understood. In a previous report, we developed color-coded imaging of the tumor microenvironment (TME) of the murine EL4-RFP malignant lymphoma during metastasis, including the lung. In the present report, we investigated the potential and microenvironment of the fatty liver induced by a choline-deficient diet as a metastatic site in this mouse lymphoma model. MATERIALS AND METHODS: C57BL/6-GFP transgenic mice were fed with a choline-deficient diet in order to establish a fatty liver model. EL4-RFP cells were injected in the spleen of normal mice and fatty-liver mice. Metastases in mice with fatty liver or normal liver were imaged with the Olympus SZX7 microscope and the Olympus FV1000 confocal microscope. RESULTS: Metastases of EL4-RFP were observed in the liver, ascites and bone marrow. Primary tumors were imaged in the spleen at the injection site. The fewest metastases were observed in the fatty liver. In addition, the fewest cancer-associated fibroblasts (CAFs) were observed in the fatty liver. CONCLUSION: The relative metastatic resistance of the fatty liver may be due to the reduced number of CAFs in the fatty livers. The mechanism of the effect of the choline-deficient diet is discussed.

Imaging the Role of Multinucleate Pancreatic Cancer Cells and Cancer-Associated Fibroblasts in Peritoneal Metastasis in Mouse Models.

BACKGROUND/AIM: The interaction between pancreatic-cancer cells and stromal cells in the tumor microenvironment (TME) is of particular importance in cancer progression and metastasis. The present report demonstrates the role of cancer-associated fibroblasts (CAFs) and multinucleate pancreatic-cancer cells in peritoneal metastasis. MATERIALS AND METHODS: An orthotopic mouse model of pancreatic cancer was established with the human pancreatic cancer cell line BxPC3, which stably expresses green fluorescent protein (GFP). RESULTS: BxPC3-GFP cells formed peritoneal metastases by week 18 after orthotopic implantation. Using an Olympus FV1000 confocal microscope, multi-nucleated cancer cells were frequently observed in the peritoneal metastases. The primary pancreatic tumor and peritoneal-metastases were harvested, cultured and then transplanted subcutaneously. Subcutaneous tumors established from peritoneal-metastatic cells were larger than subcutaneous tumors established from primary-tumor cells. Subcutaneous tumors of each type were subsequently cultured in vitro. CAFs were observed growing out from the tumors established from peritoneal-metastatic cells, but not the tumors established from the primary cancer. CONCLUSION: The results of the present study suggest that multi-nucleated cancer cells and CAFs were related to peritoneal metastasis of pancreatic cancer.

Genetic Recombination Between Stromal and Cancer Cells Results in Highly Malignant Cells Identified by Color-Coded Imaging in a Mouse Lymphoma Model.

The tumor microenvironment (TME) promotes tumor growth and metastasis. We previously established the color-coded EL4 lymphoma TME model with red fluorescent protein (RFP) expressing EL4 implanted in transgenic C57BL/6 green fluorescent protein (GFP) mice. Color-coded imaging of the lymphoma TME suggested an important role of stromal cells in lymphoma progression and metastasis. In the present study, we used color-coded imaging of RFP-lymphoma cells and GFP stromal cells to identify yellow-fluorescent genetically recombinant cells appearing only during metastasis. The EL4-RFP lymphoma cells were injected subcutaneously in C57BL/6-GFP transgenic mice and formed subcutaneous tumors 14 days after cell transplantation. The subcutaneous tumors were harvested and transplanted to the abdominal cavity of nude mice. Metastases to the liver, perigastric lymph node, ascites, bone marrow, and primary tumor were imaged. In addition to EL4-RFP cells and GFP-host cells, genetically recombinant yellow-fluorescent cells, were observed only in the ascites and bone marrow. These results indicate genetic exchange between the stromal and cancer cells. Possible mechanisms of genetic exchange are discussed as well as its ramifications for metastasis. J. Cell. Biochem. 118: 4216-4221, 2017. © 2017 Wiley Periodicals, Inc.

The Number of Point Mutations in Induced Pluripotent Stem Cells and Nuclear Transfer Embryonic Stem Cells Depends on the Method and Somatic Cell Type Used for Their Generation.

Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells 2017;35:1189-1196.

Color-coded Imaging Enables Fluorescence-guided Surgery to Resect the Tumor Along with the Tumor Microenvironment in a Syngeneic Mouse Model of EL-4 Lymphoma.

BACKGROUND/AIM: Fluorescence-guided surgery (FGS) of cancer is an emerging technology. We have previously shown the importance of resecting both the tumor and the tumor microenvironment (TME) for curative FGS. We also previously developed a syngeneic model using the mouse lymphoma cell line EL-4, expressing red fluorescent protein (EL-4-RFP), growing in green fluorescent protein (GFP) transgenic mice, which we have used in the present report to develop FGS of the tumor microenvironment. MATERIALS AND METHODS: EL-4-RFP lymphoma cells were injected subcutaneously in C57/BL6 GFP transgenic mice. EL-4-RFP cells subsequently formed tumors by 35 days after cell transplantation. Using the portable hand-held Dino-Lite digital imaging system, subcutaneous tumors were resected by FGS. Resected tumor tissues were visualized with the Olympus FV1000 confocal microscope. RESULTS: Using the Dino-Lite, subcutaneous tumors and the tumor microenvironment were clearly visualized and resected. In the resected tumor, host stromal cells, including adipocyte-like cells and blood vessels with lymphocytes, were observed by confocal microscopy in addition to cancer cells by color-coded confocal imaging. The cancer cells and stromal cells in the TME were deeply intermingled in a highly-complex pattern. CONCLUSION: Color-coded FGS is an effective method to completely resect cancer cells along with the stromal cells in the TME which interact in a highly-complex pattern. Microscopically, cancer cells invade the TME and vice versa. To prevent tumor recurrence, it is necessary to resect the TME along with the tumor.