Measurement of tumor size 

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... (s.c., formulation with or without silver, 1.0 and 3.3 mg/kg equivalent CDDP) [16]. The renal toxicity of the drug was monitored by the creatinine level in the urine of the Sprague–Dawley rats for up to 30 days following a single injection. The HA–Pt formulation with silver nitrate as an activating agent was eliminated from the study due to the renal toxicity it caused. The animals that were treated with either HA–Pt silver-free formulation or unbound CDDP did not demonstrate a statistically significant difference, which is due to the effective functional recovery of the rats following a single dose of the drug, as well as the relatively low dose that was administered. Therefore, we subsequently conducted a kidney pathological study to compare the extent of tissue degeneration and necrosis of their kidney sections. The results showed that the lymphatic HA–Pt formulation induced milder tissue damage and less renal dysfunction. At the conclusion of the 5-week toxicity study (weekly dose of 3.5 mg/kg for 3 weeks), animals were euthanized and a full pathological examination was performed. Kidney, liver, brain, lymph nodes and underlying tissue of the injection site were normal with no significant microscopic changes for all study groups. This is likely due to the low dosing level administered as well as the reduced drug uptake and accumulation or the upregulated tissue repair. Studies at a higher drug concentration, increased dosing frequency or reduced recovery time at the conclusion of the treatment may result in differentiated CDDP toxicities. In this study, we established an orthotopic murine tumor model of HNSCC by injecting human HNSCC cells – MDA-1986 – into the buccal mucosa of nude mice. The model exhibited rapid and sustained tumor growth, with average survival of up to 12 weeks after tumor cell implantation. The primary tumor proceeded similarly to highly aggressive human HNSCC, invading the mandible and metastasizing to the cervical nodes. To validate the tumor model and verify the incidence of distant metastasis, we intactly removed the primary tumor and the draining lymph nodes from the animal and stored in formalin solution overnight for fixation before biopsies. H&E staining tests were conducted and the histology slides showed muscular (middle panel) and glandular (right panel) invasion of the cancer cells into the lymphatics (Figure 1). In order for our nanocarriers to deliver anticancer drugs to metastases in the head and neck locoregional lymphatics, carriers should drain from the tumor area to the diseased lymph nodes. We injected HA–Texas Red conjugates to evaluate the drainage into the diseased cervical lymph nodes 24 h post-injection (Figure 2). The nanoconjugates slowly diffused from the injection site possibly to the primary tumor and peripheral locoregional lymphatics, which harbor the metastases. If the targeted drainage of the HA conjugates toward the neoplastic tissue occurred, it is likely to be due to the lymphangiogenesis, leading to the increased accumulation of the nanoconjugates adjacent to the tumor and the cervical lymph nodes. In order to verify the uptake, additional histological evidences for the targeted drainage using a fluorescent microscope will be conducted in our ongoing future experiments. Other polymeric carriers, such as optically labeled dendrimer [17] or dextran [18,19], have also been used to enable visualization of the lymphatic drainage. In order to visualize tumor progression in the early stages, we developed a MDA-1986 cell line expressing red fluorescent protein. Initially the Turbo635 was inserted into the pLNCX2 vector (Clontech) under control of the CMV immediate early promoter with a neomycin insert and selected with G418. Although the resulting cells had strong fluorescence in vitro , tumor implants resulted in no fluorescence, leading us to believe that the promoter was shut off in this cell line after implantation. Replacement with the Hsp70 promoter resulted in strong expression of the fluorescent reporter gene in vitro and in vivo . Two groups of female nude mice treated with saline i.v. or HA s.c. developed tumors of approximately 1000 mm 3 on average after 5 weeks, which indicated that HA does not alter the natural progression of HNSCC. On the other hand, the female animals that were treated with three doses of either i.v. CDDP or i.v. HA–Pt developed tumors of approximately 1000 mm 3 8 weeks post-tumor cell implantation. By contrast, 57% of the female animals bearing HNSCC xenografts in the HA–Pt s.c. treated group (three equivalent doses of HA–Pt) were cured within 6 weeks with no disease recurrence by the end of the study (Figure 3). In addition, survival rate was greatly improved for the animals treated with HA–Pt s.c. when compared with either of the control groups (p = 0.0019 for saline i.v. group and p = 0.0027 for HA s.c. group) or CDDP i.v. (p = 0.0221) and HA–Pt i.v. (p = 0.0112) groups using Gehan–Breslow–Wilcoxon rank analysis (Figure 3). No statistically significant differences were observed for i.v.-treated CDDP and HA–Pt groups. Overall, the result of the tumor model suggests that HA–Pt conjugates achieved a higher anticancer efficacy relative to the conventional i.v. CDDP therapy due to the route of drug administration as well as the intrinsic properties of HA as a carrier for lymphatic drug delivery (Figure 3). HA–Pt slowly released the active form of the drug, which subsequently drained to the adjacent cervical lymph nodes and the surrounding lymphatic regions. The gradually released CDDP did not cause any skin or tissue reaction at the injection site, even though CDDP is well known as a vesicant. This is likely due to the protective effect of HA, which has been used as a rescue medication to alleviate local toxicity effects of chemotherapeutics. A similar tumor suppression effect was also observed by Chen et al. in their treatment efficacy study using a CDDP-incorporated polymer conjugate against HNSCC in a chimeric mouse model [15]. Some effect may be due to the increased residence time of CDDP in plasma with the locally injected conjugates. However, this effect would be due to vascular uptake and subsequent extravasculation followed by lymphatic uptake in the head and neck region. In another study we recently published, levels in ipsilateral axilla nodes were compared with contralateral, and drug levels in the contralateral were similar to intravenous levels, with no local ‘boost’ in even these immediate-area lymphatics with a different drainage basin [16]. Administration of anticancer drugs via polymeric delivery vehicles is a promising method for local delivery of concentrated chemotherapeutics, effectively treating lymphatically metastatic cancers. A study by Dunne et al. revealed that CDDP-bound poly(ethylene oxide)-block-poly(lysine) block copolymer greatly hindered the tumor progression in a squamous cell carcinoma model of the upper aerodigestive tract in animals [20]. Another study carried out by Xie et al. reported an enhanced efficacy against laryngeal squamous cell carcinoma in rodents compared with conventional paclitaxel therapy [21]. Another interesting observation was that the tumor progression of HNSCC exhibited a more aggressive pattern in male nude mice compared with the female animals. All the male animals in the CDDP i.v. group reached an average tumor size of approximately 1000 mm 3 in 2 weeks (Figure 4) as opposed to 8 weeks, as observed in the previous female animal studies (Figure 3). On the other hand, 80% of the male animals in the HA–Pt s.c. group developed tumors of approximately 1000 mm 3 in 3 weeks and 20% of the animals were able to live through the end of the fourth week (Figure 4). Therefore, gender differences, as well as weight loss and stress, may be contributing factors that are responsible for the differentiated carcinoma progression. In addition, gender differences in the pharmacokinetics and tolerability of CDDP may also impact the differences in survival. Stakisaitis et al . found in rats that CDDP-related hyponatermia and renal toxicity is more pronounced in males than females, although the mechanism is not understood [22]. In human studies, tobacco and alcohol consumption were believed to be the major, but not the only, factors inducing the occurrence and recurrence of HNSCC. Other factors, such as age, weight loss, nutritional status and complications may also play a role in the survival of patients [23]. Dahlstrom et al. conducted a demographic study of 172 never smoker–never drinker patients, describing the age and sex distribution of HNSCC patients, and identifying the specific types of most commonly diagnosed HNSCC [24]. A similar study was also performed by Onyango et al ., showing an overall male preponderance in the occurrence of HNSCC among 793 patience [25]. Additional factors affecting the disease progression and metastasis are still under investigation. This study demonstrates that intralymphatic delivery of CDDP may be a promising treatment regimen to deliver chemotherapeutics to the primary malignancy, locoregional lymphatics and metastases, with greatly improved in vivo efficacy and survival compared with conventional CDDP chemotherapy. Successful completion of this study may provide a platform for the development of other polymeric drug-delivery models for the treatment of a wide spectrum of lymphatically metastatic cancers. The platinum-based HA conjugates can either be administered as a neoadjuvant therapy prior to surgery to reduce tumor size and control cancer progression, or given as an adjuvant therapy post-surgery to reduce the risk of recurrence and eradicate cancer residuals, such as micrometastases. In addition, due to the sustained release characteristics of the conjugate, weekly or biweekly HA–Pt injections could replace the conventional daily infusion, leading to improved patient compliance and reduced healthcare cost. Despite major strides in ...

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... Thus, an increased dose in our treatment regimen may be expected to induce a noticeable change in the ototoxicity. Renal toxicity of CDDP is another dose-limiting side effect of the anticancer drug. In one of our previous studies, we examined the renal toxicity induced by the free CDDP (i.v., 1.0 and 3.3 mg/kg) or HA–Pt (s.c., formulation with or without silver, 1.0 and 3.3 mg/kg equivalent CDDP) [16]. The renal toxicity of the drug was monitored by the creatinine level in the urine of the Sprague–Dawley rats for up to 30 days following a single injection. The HA–Pt formulation with silver nitrate as an activating agent was eliminated from the study due to the renal toxicity it caused. The animals that were treated with either HA–Pt silver-free formulation or unbound CDDP did not demonstrate a statistically significant difference, which is due to the effective functional recovery of the rats following a single dose of the drug, as well as the relatively low dose that was administered. Therefore, we subsequently conducted a kidney pathological study to compare the extent of tissue degeneration and necrosis of their kidney sections. The results showed that the lymphatic HA–Pt formulation induced milder tissue damage and less renal dysfunction. At the conclusion of the 5-week toxicity study (weekly dose of 3.5 mg/kg for 3 weeks), animals were euthanized and a full pathological examination was performed. Kidney, liver, brain, lymph nodes and underlying tissue of the injection site were normal with no significant microscopic changes for all study groups. This is likely due to the low dosing level administered as well as the reduced drug uptake and accumulation or the upregulated tissue repair. Studies at a higher drug concentration, increased dosing frequency or reduced recovery time at the conclusion of the treatment may result in differentiated CDDP toxicities. In this study, we established an orthotopic murine tumor model of HNSCC by injecting human HNSCC cells – MDA-1986 – into the buccal mucosa of nude mice. The model exhibited rapid and sustained tumor growth, with average survival of up to 12 weeks after tumor cell implantation. The primary tumor proceeded similarly to highly aggressive human HNSCC, invading the mandible and metastasizing to the cervical nodes. To validate the tumor model and verify the incidence of distant metastasis, we intactly removed the primary tumor and the draining lymph nodes from the animal and stored in formalin solution overnight for fixation before biopsies. H&E staining tests were conducted and the histology slides showed muscular (middle panel) and glandular (right panel) invasion of the cancer cells into the lymphatics (Figure 1). In order for our nanocarriers to deliver anticancer drugs to metastases in the head and neck locoregional lymphatics, carriers should drain from the tumor area to the diseased lymph nodes. We injected HA–Texas Red conjugates to evaluate the drainage into the diseased cervical lymph nodes 24 h post-injection (Figure 2). The nanoconjugates slowly diffused from the injection site possibly to the primary tumor and peripheral locoregional lymphatics, which harbor the metastases. If the targeted drainage of the HA conjugates toward the neoplastic tissue occurred, it is likely to be due to the lymphangiogenesis, leading to the increased accumulation of the nanoconjugates adjacent to the tumor and the cervical lymph nodes. In order to verify the uptake, additional histological evidences for the targeted drainage using a fluorescent microscope will be conducted in our ongoing future experiments. Other polymeric carriers, such as optically labeled dendrimer [17] or dextran [18,19], have also been used to enable visualization of the lymphatic drainage. In order to visualize tumor progression in the early stages, we developed a MDA-1986 cell line expressing red fluorescent protein. Initially the Turbo635 was inserted into the pLNCX2 vector (Clontech) under control of the CMV immediate early promoter with a neomycin insert and selected with G418. Although the resulting cells had strong fluorescence in vitro , tumor implants resulted in no fluorescence, leading us to believe that the promoter was shut off in this cell line after implantation. Replacement with the Hsp70 promoter resulted in strong expression of the fluorescent reporter gene in vitro and in vivo . Two groups of female nude mice treated with saline i.v. or HA s.c. developed tumors of approximately 1000 mm 3 on average after 5 weeks, which indicated that HA does not alter the natural progression of HNSCC. On the other hand, the female animals that were treated with three doses of either i.v. CDDP or i.v. HA–Pt developed tumors of approximately 1000 mm 3 8 weeks post-tumor cell implantation. By contrast, 57% of the female animals bearing HNSCC xenografts in the HA–Pt s.c. treated group (three equivalent doses of HA–Pt) were cured within 6 weeks with no disease recurrence by the end of the study (Figure 3). In addition, survival rate was greatly improved for the animals treated with HA–Pt s.c. when compared with either of the control groups (p = 0.0019 for saline i.v. group and p = 0.0027 for HA s.c. group) or CDDP i.v. (p = 0.0221) and HA–Pt i.v. (p = 0.0112) groups using Gehan–Breslow–Wilcoxon rank analysis (Figure 3). No statistically significant differences were observed for i.v.-treated CDDP and HA–Pt groups. Overall, the result of the tumor model suggests that HA–Pt conjugates achieved a higher anticancer efficacy relative to the conventional i.v. CDDP therapy due to the route of drug administration as well as the intrinsic properties of HA as a carrier for lymphatic drug delivery (Figure 3). HA–Pt slowly released the active form of the drug, which subsequently drained to the adjacent cervical lymph nodes and the surrounding lymphatic regions. The gradually released CDDP did not cause any skin or tissue reaction at the injection site, even though CDDP is well known as a vesicant. This is likely due to the protective effect of HA, which has been used as a rescue medication to alleviate local toxicity effects of chemotherapeutics. A similar tumor suppression effect was also observed by Chen et al. in their treatment efficacy study using a CDDP-incorporated polymer conjugate against HNSCC in a chimeric mouse model [15]. Some effect may be due to the increased residence time of CDDP in plasma with the locally injected conjugates. However, this effect would be due to vascular uptake and subsequent extravasculation followed by lymphatic uptake in the head and neck region. In another study we recently published, levels in ipsilateral axilla nodes were compared with contralateral, and drug levels in the contralateral were similar to intravenous levels, with no local ‘boost’ in even these immediate-area lymphatics with a different drainage basin [16]. Administration of anticancer drugs via polymeric delivery vehicles is a promising method for local delivery of concentrated chemotherapeutics, effectively treating lymphatically metastatic cancers. A study by Dunne et al. revealed that CDDP-bound poly(ethylene oxide)-block-poly(lysine) block copolymer greatly hindered the tumor progression in a squamous cell carcinoma model of the upper aerodigestive tract in animals [20]. Another study carried out by Xie et al. reported an enhanced efficacy against laryngeal squamous cell carcinoma in rodents compared with conventional paclitaxel therapy [21]. Another interesting observation was that the tumor progression of HNSCC exhibited a more aggressive pattern in male nude mice compared with the female animals. All the male animals in the CDDP i.v. group reached an average tumor size of approximately 1000 mm 3 in 2 weeks (Figure 4) as opposed to 8 weeks, as observed in the previous female animal studies (Figure 3). On the other hand, 80% of the male animals in the HA–Pt s.c. group developed tumors of approximately 1000 mm 3 in 3 weeks and 20% of the animals were able to live through the end of the fourth week (Figure 4). Therefore, gender differences, as well as weight loss and stress, may be contributing factors that are responsible for the differentiated carcinoma progression. In addition, gender differences in the pharmacokinetics and tolerability of CDDP may also impact the differences in survival. Stakisaitis et al . found in rats that CDDP-related hyponatermia and renal toxicity is more pronounced in males than females, although the mechanism is not understood [22]. In human studies, tobacco and alcohol consumption were believed to be the major, but not the only, factors inducing the occurrence and recurrence of HNSCC. Other factors, such as age, weight loss, nutritional status and complications may also play a role in the survival of patients [23]. Dahlstrom et al. conducted a demographic study of 172 never smoker–never drinker patients, describing the age and sex distribution of HNSCC patients, and identifying the specific types of most commonly diagnosed HNSCC [24]. A similar study was also performed by Onyango et al ., showing an overall male preponderance in the occurrence of HNSCC among 793 patience [25]. Additional factors affecting the disease progression and metastasis are still under investigation. This study demonstrates that intralymphatic delivery of CDDP may be a promising treatment regimen to deliver chemotherapeutics to the primary malignancy, locoregional lymphatics and metastases, with greatly improved in vivo efficacy and survival compared with conventional CDDP chemotherapy. Successful completion of this study may provide a platform for the development of other polymeric drug-delivery models for the treatment of a wide spectrum of lymphatically metastatic cancers. The platinum-based HA conjugates can either be administered as a neoadjuvant therapy prior to surgery to reduce tumor size and control cancer progression, or given as an adjuvant therapy post-surgery to reduce the risk of ...

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... that were treated with either HA–Pt silver-free formulation or unbound CDDP did not demonstrate a statistically significant difference, which is due to the effective functional recovery of the rats following a single dose of the drug, as well as the relatively low dose that was administered. Therefore, we subsequently conducted a kidney pathological study to compare the extent of tissue degeneration and necrosis of their kidney sections. The results showed that the lymphatic HA–Pt formulation induced milder tissue damage and less renal dysfunction. At the conclusion of the 5-week toxicity study (weekly dose of 3.5 mg/kg for 3 weeks), animals were euthanized and a full pathological examination was performed. Kidney, liver, brain, lymph nodes and underlying tissue of the injection site were normal with no significant microscopic changes for all study groups. This is likely due to the low dosing level administered as well as the reduced drug uptake and accumulation or the upregulated tissue repair. Studies at a higher drug concentration, increased dosing frequency or reduced recovery time at the conclusion of the treatment may result in differentiated CDDP toxicities. In this study, we established an orthotopic murine tumor model of HNSCC by injecting human HNSCC cells – MDA-1986 – into the buccal mucosa of nude mice. The model exhibited rapid and sustained tumor growth, with average survival of up to 12 weeks after tumor cell implantation. The primary tumor proceeded similarly to highly aggressive human HNSCC, invading the mandible and metastasizing to the cervical nodes. To validate the tumor model and verify the incidence of distant metastasis, we intactly removed the primary tumor and the draining lymph nodes from the animal and stored in formalin solution overnight for fixation before biopsies. H&E staining tests were conducted and the histology slides showed muscular (middle panel) and glandular (right panel) invasion of the cancer cells into the lymphatics (Figure 1). In order for our nanocarriers to deliver anticancer drugs to metastases in the head and neck locoregional lymphatics, carriers should drain from the tumor area to the diseased lymph nodes. We injected HA–Texas Red conjugates to evaluate the drainage into the diseased cervical lymph nodes 24 h post-injection (Figure 2). The nanoconjugates slowly diffused from the injection site possibly to the primary tumor and peripheral locoregional lymphatics, which harbor the metastases. If the targeted drainage of the HA conjugates toward the neoplastic tissue occurred, it is likely to be due to the lymphangiogenesis, leading to the increased accumulation of the nanoconjugates adjacent to the tumor and the cervical lymph nodes. In order to verify the uptake, additional histological evidences for the targeted drainage using a fluorescent microscope will be conducted in our ongoing future experiments. Other polymeric carriers, such as optically labeled dendrimer [17] or dextran [18,19], have also been used to enable visualization of the lymphatic drainage. In order to visualize tumor progression in the early stages, we developed a MDA-1986 cell line expressing red fluorescent protein. Initially the Turbo635 was inserted into the pLNCX2 vector (Clontech) under control of the CMV immediate early promoter with a neomycin insert and selected with G418. Although the resulting cells had strong fluorescence in vitro , tumor implants resulted in no fluorescence, leading us to believe that the promoter was shut off in this cell line after implantation. Replacement with the Hsp70 promoter resulted in strong expression of the fluorescent reporter gene in vitro and in vivo . Two groups of female nude mice treated with saline i.v. or HA s.c. developed tumors of approximately 1000 mm 3 on average after 5 weeks, which indicated that HA does not alter the natural progression of HNSCC. On the other hand, the female animals that were treated with three doses of either i.v. CDDP or i.v. HA–Pt developed tumors of approximately 1000 mm 3 8 weeks post-tumor cell implantation. By contrast, 57% of the female animals bearing HNSCC xenografts in the HA–Pt s.c. treated group (three equivalent doses of HA–Pt) were cured within 6 weeks with no disease recurrence by the end of the study (Figure 3). In addition, survival rate was greatly improved for the animals treated with HA–Pt s.c. when compared with either of the control groups (p = 0.0019 for saline i.v. group and p = 0.0027 for HA s.c. group) or CDDP i.v. (p = 0.0221) and HA–Pt i.v. (p = 0.0112) groups using Gehan–Breslow–Wilcoxon rank analysis (Figure 3). No statistically significant differences were observed for i.v.-treated CDDP and HA–Pt groups. Overall, the result of the tumor model suggests that HA–Pt conjugates achieved a higher anticancer efficacy relative to the conventional i.v. CDDP therapy due to the route of drug administration as well as the intrinsic properties of HA as a carrier for lymphatic drug delivery (Figure 3). HA–Pt slowly released the active form of the drug, which subsequently drained to the adjacent cervical lymph nodes and the surrounding lymphatic regions. The gradually released CDDP did not cause any skin or tissue reaction at the injection site, even though CDDP is well known as a vesicant. This is likely due to the protective effect of HA, which has been used as a rescue medication to alleviate local toxicity effects of chemotherapeutics. A similar tumor suppression effect was also observed by Chen et al. in their treatment efficacy study using a CDDP-incorporated polymer conjugate against HNSCC in a chimeric mouse model [15]. Some effect may be due to the increased residence time of CDDP in plasma with the locally injected conjugates. However, this effect would be due to vascular uptake and subsequent extravasculation followed by lymphatic uptake in the head and neck region. In another study we recently published, levels in ipsilateral axilla nodes were compared with contralateral, and drug levels in the contralateral were similar to intravenous levels, with no local ‘boost’ in even these immediate-area lymphatics with a different drainage basin [16]. Administration of anticancer drugs via polymeric delivery vehicles is a promising method for local delivery of concentrated chemotherapeutics, effectively treating lymphatically metastatic cancers. A study by Dunne et al. revealed that CDDP-bound poly(ethylene oxide)-block-poly(lysine) block copolymer greatly hindered the tumor progression in a squamous cell carcinoma model of the upper aerodigestive tract in animals [20]. Another study carried out by Xie et al. reported an enhanced efficacy against laryngeal squamous cell carcinoma in rodents compared with conventional paclitaxel therapy [21]. Another interesting observation was that the tumor progression of HNSCC exhibited a more aggressive pattern in male nude mice compared with the female animals. All the male animals in the CDDP i.v. group reached an average tumor size of approximately 1000 mm 3 in 2 weeks (Figure 4) as opposed to 8 weeks, as observed in the previous female animal studies (Figure 3). On the other hand, 80% of the male animals in the HA–Pt s.c. group developed tumors of approximately 1000 mm 3 in 3 weeks and 20% of the animals were able to live through the end of the fourth week (Figure 4). Therefore, gender differences, as well as weight loss and stress, may be contributing factors that are responsible for the differentiated carcinoma progression. In addition, gender differences in the pharmacokinetics and tolerability of CDDP may also impact the differences in survival. Stakisaitis et al . found in rats that CDDP-related hyponatermia and renal toxicity is more pronounced in males than females, although the mechanism is not understood [22]. In human studies, tobacco and alcohol consumption were believed to be the major, but not the only, factors inducing the occurrence and recurrence of HNSCC. Other factors, such as age, weight loss, nutritional status and complications may also play a role in the survival of patients [23]. Dahlstrom et al. conducted a demographic study of 172 never smoker–never drinker patients, describing the age and sex distribution of HNSCC patients, and identifying the specific types of most commonly diagnosed HNSCC [24]. A similar study was also performed by Onyango et al ., showing an overall male preponderance in the occurrence of HNSCC among 793 patience [25]. Additional factors affecting the disease progression and metastasis are still under investigation. This study demonstrates that intralymphatic delivery of CDDP may be a promising treatment regimen to deliver chemotherapeutics to the primary malignancy, locoregional lymphatics and metastases, with greatly improved in vivo efficacy and survival compared with conventional CDDP chemotherapy. Successful completion of this study may provide a platform for the development of other polymeric drug-delivery models for the treatment of a wide spectrum of lymphatically metastatic cancers. The platinum-based HA conjugates can either be administered as a neoadjuvant therapy prior to surgery to reduce tumor size and control cancer progression, or given as an adjuvant therapy post-surgery to reduce the risk of recurrence and eradicate cancer residuals, such as micrometastases. In addition, due to the sustained release characteristics of the conjugate, weekly or biweekly HA–Pt injections could replace the conventional daily infusion, leading to improved patient compliance and reduced healthcare cost. Despite major strides in cancer prevention and treatment during recent decades, there has been only a modest improvement in overall survival. Over half of HNSCC patients will face recurrence at some point, and these cancers recur because current imaging tools fail to detect occult disease or therapies fail to completely eradicate resistant disease. More frequent and intense radioimaging or stronger regimens of ...

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... drain from the tumor area to the diseased lymph nodes. We injected HA–Texas Red conjugates to evaluate the drainage into the diseased cervical lymph nodes 24 h post-injection (Figure 2). The nanoconjugates slowly diffused from the injection site possibly to the primary tumor and peripheral locoregional lymphatics, which harbor the metastases. If the targeted drainage of the HA conjugates toward the neoplastic tissue occurred, it is likely to be due to the lymphangiogenesis, leading to the increased accumulation of the nanoconjugates adjacent to the tumor and the cervical lymph nodes. In order to verify the uptake, additional histological evidences for the targeted drainage using a fluorescent microscope will be conducted in our ongoing future experiments. Other polymeric carriers, such as optically labeled dendrimer [17] or dextran [18,19], have also been used to enable visualization of the lymphatic drainage. In order to visualize tumor progression in the early stages, we developed a MDA-1986 cell line expressing red fluorescent protein. Initially the Turbo635 was inserted into the pLNCX2 vector (Clontech) under control of the CMV immediate early promoter with a neomycin insert and selected with G418. Although the resulting cells had strong fluorescence in vitro , tumor implants resulted in no fluorescence, leading us to believe that the promoter was shut off in this cell line after implantation. Replacement with the Hsp70 promoter resulted in strong expression of the fluorescent reporter gene in vitro and in vivo . Two groups of female nude mice treated with saline i.v. or HA s.c. developed tumors of approximately 1000 mm 3 on average after 5 weeks, which indicated that HA does not alter the natural progression of HNSCC. On the other hand, the female animals that were treated with three doses of either i.v. CDDP or i.v. HA–Pt developed tumors of approximately 1000 mm 3 8 weeks post-tumor cell implantation. By contrast, 57% of the female animals bearing HNSCC xenografts in the HA–Pt s.c. treated group (three equivalent doses of HA–Pt) were cured within 6 weeks with no disease recurrence by the end of the study (Figure 3). In addition, survival rate was greatly improved for the animals treated with HA–Pt s.c. when compared with either of the control groups (p = 0.0019 for saline i.v. group and p = 0.0027 for HA s.c. group) or CDDP i.v. (p = 0.0221) and HA–Pt i.v. (p = 0.0112) groups using Gehan–Breslow–Wilcoxon rank analysis (Figure 3). No statistically significant differences were observed for i.v.-treated CDDP and HA–Pt groups. Overall, the result of the tumor model suggests that HA–Pt conjugates achieved a higher anticancer efficacy relative to the conventional i.v. CDDP therapy due to the route of drug administration as well as the intrinsic properties of HA as a carrier for lymphatic drug delivery (Figure 3). HA–Pt slowly released the active form of the drug, which subsequently drained to the adjacent cervical lymph nodes and the surrounding lymphatic regions. The gradually released CDDP did not cause any skin or tissue reaction at the injection site, even though CDDP is well known as a vesicant. This is likely due to the protective effect of HA, which has been used as a rescue medication to alleviate local toxicity effects of chemotherapeutics. A similar tumor suppression effect was also observed by Chen et al. in their treatment efficacy study using a CDDP-incorporated polymer conjugate against HNSCC in a chimeric mouse model [15]. Some effect may be due to the increased residence time of CDDP in plasma with the locally injected conjugates. However, this effect would be due to vascular uptake and subsequent extravasculation followed by lymphatic uptake in the head and neck region. In another study we recently published, levels in ipsilateral axilla nodes were compared with contralateral, and drug levels in the contralateral were similar to intravenous levels, with no local ‘boost’ in even these immediate-area lymphatics with a different drainage basin [16]. Administration of anticancer drugs via polymeric delivery vehicles is a promising method for local delivery of concentrated chemotherapeutics, effectively treating lymphatically metastatic cancers. A study by Dunne et al. revealed that CDDP-bound poly(ethylene oxide)-block-poly(lysine) block copolymer greatly hindered the tumor progression in a squamous cell carcinoma model of the upper aerodigestive tract in animals [20]. Another study carried out by Xie et al. reported an enhanced efficacy against laryngeal squamous cell carcinoma in rodents compared with conventional paclitaxel therapy [21]. Another interesting observation was that the tumor progression of HNSCC exhibited a more aggressive pattern in male nude mice compared with the female animals. All the male animals in the CDDP i.v. group reached an average tumor size of approximately 1000 mm 3 in 2 weeks (Figure 4) as opposed to 8 weeks, as observed in the previous female animal studies (Figure 3). On the other hand, 80% of the male animals in the HA–Pt s.c. group developed tumors of approximately 1000 mm 3 in 3 weeks and 20% of the animals were able to live through the end of the fourth week (Figure 4). Therefore, gender differences, as well as weight loss and stress, may be contributing factors that are responsible for the differentiated carcinoma progression. In addition, gender differences in the pharmacokinetics and tolerability of CDDP may also impact the differences in survival. Stakisaitis et al . found in rats that CDDP-related hyponatermia and renal toxicity is more pronounced in males than females, although the mechanism is not understood [22]. In human studies, tobacco and alcohol consumption were believed to be the major, but not the only, factors inducing the occurrence and recurrence of HNSCC. Other factors, such as age, weight loss, nutritional status and complications may also play a role in the survival of patients [23]. Dahlstrom et al. conducted a demographic study of 172 never smoker–never drinker patients, describing the age and sex distribution of HNSCC patients, and identifying the specific types of most commonly diagnosed HNSCC [24]. A similar study was also performed by Onyango et al ., showing an overall male preponderance in the occurrence of HNSCC among 793 patience [25]. Additional factors affecting the disease progression and metastasis are still under investigation. This study demonstrates that intralymphatic delivery of CDDP may be a promising treatment regimen to deliver chemotherapeutics to the primary malignancy, locoregional lymphatics and metastases, with greatly improved in vivo efficacy and survival compared with conventional CDDP chemotherapy. Successful completion of this study may provide a platform for the development of other polymeric drug-delivery models for the treatment of a wide spectrum of lymphatically metastatic cancers. The platinum-based HA conjugates can either be administered as a neoadjuvant therapy prior to surgery to reduce tumor size and control cancer progression, or given as an adjuvant therapy post-surgery to reduce the risk of recurrence and eradicate cancer residuals, such as micrometastases. In addition, due to the sustained release characteristics of the conjugate, weekly or biweekly HA–Pt injections could replace the conventional daily infusion, leading to improved patient compliance and reduced healthcare cost. Despite major strides in cancer prevention and treatment during recent decades, there has been only a modest improvement in overall survival. Over half of HNSCC patients will face recurrence at some point, and these cancers recur because current imaging tools fail to detect occult disease or therapies fail to completely eradicate resistant disease. More frequent and intense radioimaging or stronger regimens of chemotherapy and radiation would be detrimental in the long run to patients’ health and, therefore, new approaches are needed. Localized therapies stand a stronger chance of eradicating residual disease since higher doses of chemotherapy can be administered without dose-limiting toxicity to the heart, kidneys and liver. Localized chemotherapy is already used to great success in the treament of peritoneal disease in colon cancer using heated intraperitoneal chemotherapy, limb melanomas with heated isolated limb perfusions and liver cancer using isolated limb perfusions. However, there is no way to isolate most organs and tissues for concentrated chemotherapy, such as the breasts, neck and lungs. For most tissues, the lymphatics are an ideal pathway for local therapy due to their importance in early cancer metastasis. Localized chemotherapy may become an important component in the treatment of early-stage cancers that are still confined to the primary tumor and local lymphatics; yet, to date, there are no tools in the clinic to localize chemotherapy in the lymph system. Localized delivery systems such as we have described can effectively treat these early-stage diseases and we believe they will represent a new treatment option in the next 5–10 years. Nextgeneration systems will incorporate both imaging and diagnostic capabilities in the drug or drug carriers, so that the staging (or restaging) of the disease can be followed immediately by treatment to reduce treatment costs, limit the need for invasive surgeries and intravenous treatments and increase the likelihood that all disease can be eliminated in the early, more controllable ...