Natural seaweed polysaccharide; Biochemical reagent

Effects of λ-carrageenan on cell viability of B16-F10 and 4T1 in vitro [1]
As the intratumoral injection of λ-carrageenan had a significant antitumor effect in mice, we further investigated that whether the tumor growth inhibition was induced by the direct cytotoxicity of λ-carrageenan. After the incubation of tumor cells with λ-carrageenan in vitro, the cell morphologies were recorded by microscopy and the cell viability was assessed by trypan blue exclusion test. B16-F10 and 4T1 cells were incubated with λ-carrageenan at a concentration of 0.25–1.0 mg/ml for 24 h and the cell morphologies were identified normal (Fig. 2A). Trypan blue exclusion test showed that even at the high concentration of 1.0 mg/ml, cell viabilities maintained above 80% (Fig. 2B), which indicated a low cytotoxicity of λ-carrageenan to tumor cells. Furthermore, the results of MTT cell proliferation assay had similar results (Fig. 2C). The incubation of tumor cells with λ-carrageenan showed no effect to cell proliferation after 24 h of treatment. After exposure to λ-carrageenan for 48 h, the relative cell viability decreased a little as the concentration of λ-carrageenan increased. Thus, the results suggested that λ-carrageenan had low cytotoxicity to tumor cells in vitro.

---

λ-carrageenan treatment increases the expression of IL17A and TNF-α [1]
We also examined the proportion of T helper cell 17 (Th17) in mice spleen with staining of intracellular IL17A by flow cytometry and qRT-PCR. Th17 recruits and activates neutrophils and plays an important role in immune responses to fungi and extracellular pathogens. We found that intratumoral injection of λ-carrageenan led to an increase in Th17 cells and the increased secretion of IL17A in spleen lymphocytes (Fig. 4A), which might contribute to the stimulation of immune response in tumor-bearing mice after λ-carrageenan treatment.

λ-Carrageenan (50 mg/kg every two days; intratumoral injection) suppresses the formation of tumors in B16-F10 and 4T1-bearing mice[1]. By increasing the infiltrates of immunostimulating cells and producing more proinflammatory cytokines, λ-carrageenan significantly enhances the tumor immune response and has a notable anticancer effect when injected intratumorally[1].

---

λ-Carrageenan is a seaweed polysaccharide which has been generally used as proinflammatory agent in the basic research, however, how the immunomodulating activity of λ-carrageenan affects tumor microenvironment remains unknown. In this study, we found that intratumoral injection of λ-carrageenan could inhibit tumor growth in B16-F10 and 4T1 bearing mice and enhance tumor immune response by increasing the number of tumor-infiltrating M1 macrophages, DCs and more activated CD4(+)CD8(+) T lymphocytes in spleen. In addition, λ-carrageenan could enhance the secretion of IL17A in spleen and significantly increase the level of TNF-α in tumor, most of which was secreted by infiltrating macrophages. Moreover, λ-carrageenan exhibited an efficient adjuvant effect in OVA-based preventative and therapeutic vaccine for cancer treatment, which significantly enhanced the production of anti-OVA antibody. The toxicity analysis suggested that λ-carrageenan was with a good safety profile. Thus, λ-carrageenan might be used both as a potent antitumor agent and an efficient adjuvant in cancer immunotherapy[1].

---

λ-Carrageenan inhibits tumor growth in B16-F10 and 4T1 bearing mice [1]
To investigate how intratumoral injection of λ-carrageenan affects tumor microenvironment, we have selected melanoma B16-F10 and mammary cancer 4T1 as the models. B16-F10 cells (5 × 105 cell/mice) were injected subcutaneously in mice (Fig. 1A) and 4T1 cells (1 × 106 cell/mice) were injected subcutaneously into the right dorsal flank (Fig. 1B) or injected subcutaneously into mice fat pad of the mammary gland (Fig. 1C) to establish the tumor model. The administration started after the tumors reached the average volume of 30–40 mm3. λ-Carrageenan was injected every two days intratumoraly at a dose of 50 mg/kg and the tumor volume was recorded. The intratumoral injection of λ-carrageenan led to a significant reduction in tumor volumes while compared with normal saline groups in three tumor models (Fig. 1). We noticed that the antitumor effect of λ-carrageenan was comparable to that of Adriamycin in the 4T1 model (Fig. 1C). As calculated by the formula in the Methods (1), the inhibition rate of tumor was 56.8%, 39% and 42.7% in B16-F10 and 4T1 models respectively. Also, we noticed that λ-carrageenan had more potent antitumor effect in B16-F10 tumor model, thus, we chose B16-F10 tumor as the model to further study how λ-carrageenan affected the microenvironment in tumors.

---

Intratumoral injection of λ-carrageenan stimulates tumor immune response [1]
As λ-carrageenan showed low cytotoxicity in vitro after incubation with tumor cells, we next investigated how λ-carrageenan affected tumor microenvironment in vivo after intratumoral injection in mice. Tumor microenvironment contains many distinct cell types and the immune cells play crucial roles, such as F4/80+ macrophages and CD11c+ dendritic cells, which are important in initiation of immune response and in antigen-presenting process, respectively. We have evaluated the immune cells in tumor microenvironment after the intratumoral injection of λ-carrageenan by flow cytometry. As shown in Fig. 3A (left), the proportion of F4/80low macrophages was dramatically increased by 10 times after λ-carrageenan treatment while compared with control group. In addition, CD11c+ DCs were also increased from 0.6% to 2.4% in tumor tissues (Fig. 3A, right). These results indicated that a large number of F4/80low macrophages and DCs infiltrated into tumor tissue in respond to λ-carrageenan injection.

---

λ-carrageenan as an adjuvant to enhance OVA-based vaccine potency [1]
We have evaluated the efficiency of λ-carrageenan as an adjuvant in the use of both preventative vaccine and therapeutic vaccine in mice. In the evaluation of cancer preventative vaccine, mice were vaccinated three times with OVA with or without λ-carrageenan and then challenged with E.G7-OVA cells (3 × 106 cell/mice). The volumes of tumors were recorded. The results showed that the vaccines with λ-carrageenan added as an adjuvant significantly inhibited the tumor growth while compared with normal saline and OVA group (Fig. 5A). When it comes to the therapeutic vaccine, injection of OVA showed no therapeutic effect while compared with the control, in contrast to that, injection of OVA/λ-carrageenan inhibited tumor growth to some extent while compared with other groups (Fig. 5B). After the mice were immunized with preventative vaccine with λ-carrageenan, six out of ten mice were tumor-free for more than 40 days after tumor inoculation, while all the mice in NS group and OVA group bared tumors (Fig. 5C). One week after final immunization in mice, mice serum were collected and tested by ELISA for evaluation of total anti-OVA IgG. As shown in Fig. 5D, the antibodies generated in mice immunized with OVA/λ-carrageenan were significantly increased while compared with other two groups. The absorbance rates of samples in OVA and OVA/λ-carrageenan group were 0.35 and 0.78 respectively, which represented a four times increase of the antibody titer in OVA/λ-carrageenan group while compared to OVA group (from 2000 to 8000).

TUNEL Assay [1]
To examine the cell death in B16-F10 tumors after intratumoral injection of λ-carrageenan, paraffin sections of tumor tissue specimens were stained with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) using a commercially available TUNEL kit (Promega, Madison, WI, U.S.) according to the manufacturer’s instructions. Samples were observed under a DM 2500 fluorescence microscope

Cell viability assay in vitro [1]
Cell viability was measured using trypan-blue exclusion assay and MTT assay. B16-F10 and 4T1 cells were plated into 96-multi-well plates and cultured overnight to achieve cell adhesion, respectively. Then cells were treated with different concentrations (0.25, 0.5, 1, 2.5 mg/mL) of λ-carrageenan. After 24 and 48 h of treatment, we replaced the fluid with new culture media containing MTT (0.5 mg/mL) and incubated for 4h until purple precipitate was visible. MTT was removed and 150 μL DMSO was added to dissolve the precipitate. Absorbance was measured at 570 nm using a microplate reader. Each concentration was replicated 6 wells. The blank group and control group were set up simultaneously. For Trypan blue exclusion assay, the cells were treated as MTT assay. After λ-carrageenan treatment, the status of cells was first evaluated by microscopy. Then cells were harvested and 10 μl of cell suspension was mixed with 10 μl of 0.4% Trypan blue solution for 3 min, and both unstained (viable) and stained (dead) cells were counted by the Countstar Automated Cell Counter.

---

Flow cytometry analysis [1]
After the treatment of λ-carrageenan, immune cells prepared from spleen and tumors were stained with PE or PerCP-CD4, FITC-CD8, PE-CD69, FITC or PerCP-CD11b, PE-F4/80, PE-CD11c, FITC-IL17A, FITC-TNF-α. All the fluorophore conjugated antibodies and isotype-matched mAbs were purchased from BD Pharmingen. Tumors were scissored into small pieces, and dissociated using 1 mg/mL collagenaseI in serum free RPMI 1640 medium for 2 hours. Resulting cell suspensions were centrifuged, washed in PBS and passed through a BD Falcon™ 70-μm nylon cell strainer to remove clumps of cells and debris. For cell surface staining, cells were stained with antibodies on ice for 30 min in the dark; for intracellular cytokine staining, cells were fixed/permeabilized with paraformaldehyde and Triton-X100 and then stained with intracellular antibodies overnight. Analyses were carried out on a FACS Calibur flow cytometer and data were analyzed using FlowJo software.

Tumor models and λ-carrageenan treatment λ-Carrageenan was purchased from Sigma-Aldrich (cat.22049). B16-F10 cells (5×105 cell/mice) were injected subcutaneously in the right dorsal flank of C57BL/6 mice. 4T1 cells (1×106 cell/mice) were injected subcutaneously into the right dorsal flank or injected subcutaneously into mice fat pad of the mammary gland of BALB/c mice to establish the tumor model. The administration started after the tumors reached the average volume of 30–40 mm3. λ-carrageenan (1%) dissolved in saline was injected intratumoraly in a volume of 100 μL every two days and the negative control were injected with 100 μL of normal saline (NS). Adriamycin (Melone Pharmaceutical Co., Ltd, DaLian, China) was used as an anti-tumor agent for positive control. The mice were treated with adriamycin (5 mg/kg) via intraperitoneal injection every three days. Tumor growth was evaluated by measurement of tumor diameters using a caliper every 3 days and tumor volume was calculated as (long diameter) × (short diameter)2 × 0.52. The mice were sacrificed two days after final administration and tumors and organs were harvested, weighed and fixed in 4% paraformaldehyde for histochemistry; another part of tumors were stored in liquid nitrogen for further use. Inhibition ratio was calculated by following formula: Inhibition ratio (%) = [(A−B)/A]×100, where A represents the average tumor weight of the negative control, and B represents that of the λ-carrageenan treated group.

---

Antitumor immunity experiment of OVA and λ-carrageenan For the preventative tumor vaccine experiment, the left dorsal flank of C57BL/6 mice (n = 8/group) were vaccinated s.c. 3 times (at 0, 2 and 3 weeks) with 5 μg OVA protein along or OVA protein plus 100 μg λ-carrageenan as adjuvant at a total volume of 100 μL NS. One week after the last immunization, mice were challenged with E.G7-OVA cells. The tumor cells (3 × 106) were injected s.c. into the right dorsal flank in mice. The subcutaneous tumor volume was measured every three days. The tumor formation rate of mice was observed every day. For the therapeutic antitumor vaccine experiment, EG.7-OVA cells were s.c. inoculated into the right dorsal area of C57BL/6 mice. When the tumor mass became palpable (about 3 mm in length), the tumor-bearing mice were treated by 3 vaccinations with one week interval in the left dorsal area. The subcutaneous tumor volume was measured as mentioned above.

Tumor Histopathology study and toxicity evaluation[1]
In the present study, hematoxylin and eosin stainning were performed to study the tumor morphology after λ-carrageenan treatment. As shown in Fig. 6A, B16-F10 tumor tissues from the λ-carrageenan intratumoral injection group had some area of cell necrosis and significant increase of immune cell infiltration. However, tumor sections from normal saline treated mice remained normal morphology. We have also performed TUNEL assay and cleaved caspase-3 staining for evaluation of cell death in tumor sections. As shown in Fig. 6B, the positive cells for TUNEL and cleaved caspase-3 were both significantly increased after intratumoral injection of λ-carrageenan, which might contribute to the significant antitumor effect of λ-carrageenan. The tumor sections of E.G7-OVA bearing mice treated with or without therapeutic vaccines were also presented (Fig. 6C). Mice immunized with OVA/λ-carrageenan also showed increase in cell death and more immune cell infiltration in tumors.

---

Moreover, to examine potential toxicity of λ-carrageenan, vital organs in λ-carrageenan treated mice (heart, liver, spleen, lung and kidney) were collected and sections were stained with H&E for histopathological study. As shown in Fig. 6D, no significant pathologic changes were found in λ-carrageenan treated mice. In addition, no obvious toxicities were observed in the mice as determined by appearance, body weight, fecal and urinary excretion.[1]

[1]. Antitumor and Adjuvant Activity of λ-carrageenan by Stimulating Immune Response in Cancer Immunotherapy. Sci Rep. 2015;5:11062. Published 2015 Jun 22.

Moreover, intratumoral injection of λ-carrageenan led to an increase of proinflammatory IL17A (Fig. 4A), which was secreted by a subset of T helper cells, Th17 cells. Most Th17 cells can produce high levels of effector cytokines such as TNF-α and can promote antitumor immune responses by inducing the recruitment of proinflammatory immune effector cells. This also supports our conclusion that intratumoral injectioin of λ-carrageenan could enhance tumor immune response. Besides, Malley et al. have reported the role of IL17A in the immune response during vaccine application against pneumococci. Increase of Th17 cells induced by addition of adjuvants might significantly help to improve the efficiency of a vaccine. In this study, we also used λ-carrageenan as an adjuvant in both preventative vaccine and therapeutic vaccine for cancer treatment. λ-carrageenan acted as an effective adjuvant for OVA-based vaccine in OVA-expressing E.G7 lymphoma model. Also, λ-carrageenan enhanced the production of the anti-OVA antibody in mice after immunization. This indicated that mice immunized with λ-carrageenan as the adjuvant showed strong humoral responses, which might be mediated by antibodies produced by B lymphocytes. [1]
In summary, here we have provided insights into the role of λ-carrageenan used for cancer therapy both as antitumor agent and vaccine adjuvant. λ-carrageenan exhibited considerable antitumor effect via intratumoral injection and significantly improved the tumor immune response with increased infiltrates of immunostimulating cells and increased production of proinflammatory cytokines. While used as vaccine adjuvant, λ-carrageenan notably increased the efficiency of both preventative and therapeutic cancer vaccines. In addition, injection of λ-carrageenan showed no toxicities to vital organs in treated mice. Thus, we conclude that λ-carrageenan is a potent antitumor agent and efficient adjuvant which might be further used in cancer treatment to enhance tumor immune response.[1]

1139.92

9064-57-7

91972149

White to off-white solid powder

-6.1

5

20

7

35

969

0

S(=O)(=O)([O-])OC1([H])C([H])(C([H])(C([H])(C([H])([H])O[H])OC1([H])OC1([H])C([H])(C([H])([H])OS(=O)(=O)[O-])OC([H])(C([H])(C1([H])O[H])OS(=O)(=O)[O-])O[H])O[H])O[H]

UWPXLSAITSWCRB-UHFFFAOYSA-K

InChI=1S/C12H22O20S3/c13-1-3-5(14)6(15)10(32-35(24,25)26)12(29-3)30-8-4(2-27-33(18,19)20)28-11(17)9(7(8)16)31-34(21,22)23/h3-17H,1-2H2,(H,18,19,20)(H,21,22,23)(H,24,25,26)/p-3

[5-[4,5-dihydroxy-6-(hydroxymethyl)-3-sulfonatooxyoxan-2-yl]oxy-2,4-dihydroxy-6-(sulfonatooxymethyl)oxan-3-yl] sulfate

9064-57-7; UWPXLSAITSWCRB-UHFFFAOYSA-K; YC41782;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: 25 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)