The ideal cancer treatment modality should not only cause tumor regression and eradication but also induce a systemic antitumor immunity, which is essential for control of metastatic tumors and for long-term tumor resistance. Laser immunotherapy using a laser, a laser-absorbing dye, and an immunoadjuvant has induced such long-term immunity in treatment of a mammary metastatic tumor. The successfully treated rats established total resistance to multiple subsequent tumor challenges. To further study the mechanisms of the antitumor immunity induced by this novel treatment modality, passive adoptive transfer was performed using splenocytes as immune cells. The spleen cells that were harvested from successfully treated tumor-bearing rats provided 100%immunity in the naive recipients. The passively protected first cohort rats were immune to tumor challenge with an increased tumor dose; their splenocytes also prevented the establishment of tumor in the second cohort of naive recipient rats. This immunity transfer was accomplished without the usually required T-cell suppression in recipients.

The most effective cancer treatment modality should target the host immune system so that it not only can eradicate the treated primary tumors but also cause regression and eradication of metastases. Furthermore, such a modality should produce long-term resistance to the tumors to which the treated hosts were originally exposed. Tumor immunity has been achieved by immunotherapy(1, 2, 3, 4, 5, 6, 7) or by a combination of chemotherapy and immunotherapy (8, 9). Antitumor immunity resulting from using an immunoadjuvant has been shown to reject subsequent tumor challenges and to be adoptively transferred using immune lymphoid(1) or spleen cells (2, 3, 4, 5, 6, 7, 8, 9) in animal experiments. However, in most cases, such immunity transfer requires T-cell suppression in recipients, (1, 3, 4, 5, 10, 11), and the immunity often loses its resistance to tumor significantly, when sequentially and passively transferred (8). Enhanced immunity was also observed after photodynamic therapy (PDT), using light-activated photosensitizers (12, 13, 14, 15).

To induce a long-term tumor-specific immunity, a novel treatment modality, laser immunotherapy, was developed. It uses a novel immunoadjuvant administered together with a laser-absorbing dye,followed by noninvasive irradiation by a near-IR3 laser. This novel therapy caused regression of both treated primary tumors and untreated metastases in animal studies. It also induced a long-term resistance to subsequent tumor challenges. Histochemical and immunological studies showed that the laser immunotherapy treatment has induced a tumor-specific host immune response (16, 17, 18).

To test the protection ability of the induced immunity, several groups of successfully treated rats were challenged repeatedly with increased inoculation dose of the tumor cells to which they were originally exposed. To study the mechanism of the induced antitumor immunity, adoptive transfer using immune spleen cells was performed. Studied were the resistance to tumor challenges after laser immunotherapy treatment as well as the inhibition of tumor growth in naïve recipients. The protection against subsequent tumor challenge after immunity transfer was studied and the ability of the passively transferred immunity to protect subsequent cohorts of naïve recipient rats was also tested.

Tumor Model.

DMBA-4, the transplantable, metastatic mammary tumor model(19) in female Wistar Furth rats (Harlan Sprague Dawley,Indianapolis, Indiana) was used in the experiments. The tumor cells were harvested from live tumor-bearing rats and naive rats were inoculated s.c. in one of the inguinal fat pads, 7–10 days before laser immunotherapy treatment. The usual inoculation dose is 105 viable tumor cells per rat. Without treatment, the tumor-bearing rats usually survive an average of 30 days after tumor inoculation.

Laser Immunotherapy.

This novel treatment method consists of three components: a near-IR diode laser, ICG, and GC. The solution of ICG, serving as the laser-absorbing dye, and GC, serving as the immune stimulant, was directly injected into the tumor before the noninvasive laser irradiation. The injection dose was 200 μl of a solution containing 0.25% ICG and 1% GC. The tumor was irradiated with the 805-nm laser at 2 W (Continued Wave) for 10 min. The successfully treated usually experienced a gradual regression in both treated primary tumor and untreated metastases. (Procedures of ICG/GC preparation and laser immunotherapy treatment are detailed in Refs.16, 17, 18.)

Adoptive Immunization.

Viable tumor tissue was harvested from live rats bearing the DMBA-4 tumor and was dispersed to a single-cell suspension by grinding in a loose-fitting ground glass homogenizer. Those rats successfully treated by laser immunotherapy were challenged with an increased tumor dose of 106 cells per rat. At the same time, control rats were inoculated with a dose of 105 cells per rat. Twenty-eight days after the tumor rechallenge, the long-surviving rats were killed by cervical dislocation, and their spleens were dissected free of fat. Two separate experiments were conducted using the splenocytes from control tumor-bearing rats. The spleen cells were harvested 22 days and 39 days after tumor inoculation in the first and second experiment, respectively. Cell suspensions were prepared by mechanical disruption into medium with 10% FCS. The spleen cells were also collected from a naive rat without prior exposure to the tumor cells. Spleen cells and viable tumor cells were counted on a hemocytometer before admixed. The admixture had a 400:1 spleen:tumor cell ratio. Naïve rats were inoculated with the admixture containing 4 × 107spleen cells and 105 tumor cells in a volume of 200 μl.

Rechallenge of Cured Rats.

After successful treatment by laser immunotherapy, 15 cured rats were challenged with 106 viable tumor cells 120 days after the treatment. To compare with the tumor growth in control rats of the same age, 18 naïve rats of the same age (25 weeks) were inoculated with a dose of 106 viable tumor cells per rat. As shown in Table 1, all of the cured rats showed total resistance to the challenge;neither primary tumors nor metastases were observed. However, the control rats all developed primary and metastatic tumors and died around 30 days after the inoculation. The survival time appeared to be dependent on the tumor dose; the control rats with the inoculation of 105 cells survived, on average, 33 days, whereas the control rats with inoculation of 106 cells survived only 28 days.

After the first rechallenge, the rats from several different experimental groups were followed by two subsequent challenges in a time interval from 1–5 months, again with the increased tumor dose. As shown in Table 1, all of the cured rats were totally refractory to three tumor challenges after the successful treatment by laser immunotherapy.

Adoptive Immunity.

Naive rats were divided into four different groups for the adoptive immunity transfer experiments and then were inoculated with tumor cells. Group A contained the tumor-bearing control rats,inoculated by 105 viable tumor cells without any treatment. Group B contained the rats inoculated with tumor cells admixed with spleen cells from a control tumor-bearing rat. Group C contained rats inoculated with tumor cells admixed with immune spleen cells from a laser immunotherapy cured rat, 28 days after tumor rechallenge. Group D contained the rats inoculated with viable tumor cells admixed with spleen cells harvested from a naive rat without prior exposure to tumor. The experiment was performed two separate times. Fig. 1 displays the survival curves for all of the four groups from both experiments. The spleen cells from a laser immunotherapy-cured rat provided 100% protection to the recipients; neither primary nor metastatic tumors were observed among the rats in Group C. The control rats in Group A all died with multiple metastases within 35 days of tumor inoculation. The spleen cells from a healthy rat did not provide any protection to the recipients in Group D (Fig. 1, thin solid curve). Only 1 in 10 rats in Group B survived (Fig. 1, dotted curve); however, this rat developed both primary tumor (caused directly by implantation of tumor cells) and metastases.

Sixty days after the adoptive immunity transfer, all of the rats in Group C were challenged again, and all withstood the challenge. The immune spleen cells of the rats in Group C were collected and admixed with tumor cells in the same ratio as in the first adoptive transfer to test the ability of their splenocytes in protecting a subsequent cohort of normal Wistar Furth recipient rats. Six naive rats were inoculated with this admixture. The results are shown in Fig. 2, together with the results of the first protected cohort (Group C in Fig. 1, thick solid line) and of the control rats (Group A in Fig. 1, gray line). The immune cells from the rats in Group C protected five of six rats (the thin solid curve);neither primary tumor nor metastases were observed in the five surviving rats. One rat in this group died with a prolonged survival time, in comparison with the control group (60 versus 30 days), and with a delayed emergence of tumors 37 days after tumor inoculation, compared with 7–10 days in control rats.

Our previous animal experiments showed the effectiveness of laser immunotherapy in treating metastatic tumors through a local application(16, 17, 18). This novel treatment modality uses the combined effect of a selective photothermal effect and an immunological effect. The selective photothermal effect is achieved through direct application of a near-IR laser and an absorbing dye with a corresponding absorption spectrum (20). The immunological effect is achieved by introducing a novel immunoadjuvant, GC, to the treatment site. The selective photothermal reaction reduces the tumor burden and at the same time exposes the tumor antigens; the immunoadjuvant in situ first stimulates the host immune system and then directs the immune system against the residue tumor and metastases. Each individual treated host, in fact, produced an in situ autovaccine as a result. It is the tandem effect that not only resulted in total tumor eradication but also led to a long-term tumor-specific immunity. This method, therefore, provides a systemic immunotherapy for each individual host without the usually required immune cross-reactivity.

The rats that are successfully treated by laser immunotherapy can withstand subsequent challenges with the tumor dose increased several times, as shown in Table 1. The tumor challenges were performed on cured rats from four different experimental groups and at different time intervals. These results show that the induced immunity indeed has a long-lasting effect.

Other methods, such as surgery, radiation, and chemotherapy could also have curative effect on the DMBA-4 bearing rats. However, the rechallenge resistance and immunity transferability attained by these treatment modalities may not reach the potency observed with laser immunotherapy, although this remains to be experimentally verified.

Our experiments also show that the immunity can be passively transferred using immune spleen cells. After the laser immunotherapy,the immune splenocytes from cured rats can provide 100% protection to the normal recipient rats when admixed with tumor cells, as shown in Fig. 1. Apparently, the spleen cells from laser immunotherapy-treated rats totally inhibited the tumor growth; all of the rats survived, and none developed tumors. These passively protected first cohort rats are immune to tumor rechallenge, and their spleen cells can provide strong protection to a second cohort of normal recipients, as shown in Fig. 2. The protection to the second cohort of recipients reached 83%. The only nonsurvival rat in this second cohort had a long survival time (60 versus 30 days of controls) and a delayed emergence of primary tumors (37 versus 7–10 days of controls). Strong immunity has been shown to be induced by this modality of laser immunotherapy. In comparison, the spleen cells from a naïve donor not exposed to the tumor cells did not show any impact on rat survival or on tumor growth in normal recipient rats, as show by the data in Fig. 1.

The spleen cells from a donor rat bearing the same tumor provided a limited protective effect to the recipients (1 in 10 rats in the two separate experiments survived), as shown in Fig. 2. This could be attributed to the natural immune development in the host after the exposure to the tumor. However, the protection by the tumor-bearing rat spleen cells was not strong enough to inhibit the tumor growth. Even the surviving rat developed primary and metastatic tumors and then later regressed.

DMBA-4 bearing rats could produce natural immunity against the tumor. Such immunity may not be developed early enough or strongly enough to control the original tumor, as evidenced by our control experiments. However, such immunity could manifest strongly in the immune spleen cells, as evidenced by the experimental results in its adoptive transfer (see Group B in Fig. 1). Spleen cells harvested from control tumor-bearing rats may have different potencies according to the time of harvest. In the two experiments, one naive recipient rat survived after receiving spleen cells harvested from a tumor-bearing rat 39 days after inoculation, whereas no long-term survivor resulted from using spleen cells harvested 22 days after inoculation. More studies are needed to understand the development of native immune defense using spleen cells from tumor-bearing rats killed at different times.

Although passive transfer of tumor immunity has been reported after immunotherapy (1, 2, 3, 4, 5, 6, 7, 8, 9), the most successful adoptive immunity transfers required T-cell depletion in the recipients(1, 3, 4, 5, 10, 11). Furthermore, a large amount of the immune cells was often needed in the transfer (an effective ratio of 1000:1 was reported in Ref. 8), and the subsequent protection of the passive transfer could be diminished significantly. For instance, immune cells from the passively protected rats in the first cohorts could only protect 30% rats in the second cohort of naive recipients (8).

Laser immunotherapy produced a much stronger immunity based on the following findings: (a) the successfully treated rats can withstand repeated challenges with increased tumor dose;(b) the passive adoptive immunity transfer in our experiments does not require the T-cell suppression in recipients;(c) a ratio of 400:1 immune:tumor cells can provide 100%passive protection to the first cohort of naïve recipients; and(d) the spleen cells from the protected rats in the first cohort can strongly protect the second cohort of normal recipients (at an 83% level). These results may be related to the proliferation of donor cells in syngeneic hosts or to the immunological recruitment and expansion of recipient responses. Our current experiments are not able to determine which is the true cause of these phenomena.

The dose of immune cells may be an important factor in the adoptive immunity transfer, as indicated by the results in Ref. 8. As the first step in determining the protective ability, only one spleen cell:tumor cell ratio (400:1) was used in our experiments. Future studies with different ratios will yield important information on this protection ability of the immune spleen cells. Furthermore, it is important to learn which subset(s) of splenocytes is (are)responsible for the observed results. These studies are currently in progress.

Fig. 1.

Rat survival curves in the adoptive immunity experiments using rat splenocytes as immune cells. Data collected from two separate experiments were combined and plotted together. Viable tumor cells were admixed with spleen cells from different rats, then injected into naive rats. The ratio is 40,000,000 spleen cells:100,000 tumor cells per rat. Thick gray line, the rats in Group A, the tumor control rats; dotted line, the rats in Group B, injected with tumor cells admixed with spleen cells from an untreated tumor-bearing rat; thick solid line, the rats in Group C, injected with tumor cells admixed with spleen cells from a laser immunotherapy-cured rat; thin solid line, the result of using spleen cells from a naive rat (Group D). The spleen cells from the laser immunotherapy-treated rats totally inhibited the tumor growth; all of the rats survived, and none developed tumors. In comparison, only the spleen cells from tumor-bearing rats had an impact on rat survival: a 10% survival rate(dotted line). However, rats in all of the groups except in Group C developed metastases.

Fig. 1.

Rat survival curves in the adoptive immunity experiments using rat splenocytes as immune cells. Data collected from two separate experiments were combined and plotted together. Viable tumor cells were admixed with spleen cells from different rats, then injected into naive rats. The ratio is 40,000,000 spleen cells:100,000 tumor cells per rat. Thick gray line, the rats in Group A, the tumor control rats; dotted line, the rats in Group B, injected with tumor cells admixed with spleen cells from an untreated tumor-bearing rat; thick solid line, the rats in Group C, injected with tumor cells admixed with spleen cells from a laser immunotherapy-cured rat; thin solid line, the result of using spleen cells from a naive rat (Group D). The spleen cells from the laser immunotherapy-treated rats totally inhibited the tumor growth; all of the rats survived, and none developed tumors. In comparison, only the spleen cells from tumor-bearing rats had an impact on rat survival: a 10% survival rate(dotted line). However, rats in all of the groups except in Group C developed metastases.

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Fig. 2.

Rat survival rate for the second cohort of recipient rats using passive adoptive transfer. Immune cells were collected from rats protected by the first immunity transfer and admixed with tumor cells in a ratio of 400:1. The admixture was injected into a second cohort of naive recipient rats. Thin solid curve, six rats in this group; thick gray line, the tumor control rats(Group A inFig. 1); thick solid line,the results of the first adoptive immunity transfer (Group C inFig. 1). Five (83%) of six rats in the second cohort of rats showed total tumor resistance; only one rat in this group died, but with a longer survival time and delayed emergence of primary and metastatic tumors.

Fig. 2.

Rat survival rate for the second cohort of recipient rats using passive adoptive transfer. Immune cells were collected from rats protected by the first immunity transfer and admixed with tumor cells in a ratio of 400:1. The admixture was injected into a second cohort of naive recipient rats. Thin solid curve, six rats in this group; thick gray line, the tumor control rats(Group A inFig. 1); thick solid line,the results of the first adoptive immunity transfer (Group C inFig. 1). Five (83%) of six rats in the second cohort of rats showed total tumor resistance; only one rat in this group died, but with a longer survival time and delayed emergence of primary and metastatic tumors.

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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported in part by grants from the University of Central Oklahoma and from Oklahoma Center for Advancement of Science and Technology (AP00(2)-011P; PI: Wei R. Chen) and by grants from National Institute of Health (CA69043 and CA70209; PI: Hong Liu).

3

The abbreviations used are: IR, ionizing radiation; ICG, indocyanine green; GC, glycated chitosan.

Table 1

Tumor rechallenge of rats previously cured by laser immunotherapy treatment

GroupNo. of ratsNo. of tumor cellsTumor occurrence %Death rate % (in 30 days)Death rate % (in 40 days)Survival (days)
Young control ratsa 20 105 100 20 100 32.7± 3.5 
Age-matched control ratsb 18 106 100 83 100 28.2± 2.8 
Cured rats (1st challenge)c 15 106 >120 
Cured rats (2nd challenge)d 15 106 >120 
Cured rats (3rd challenge)e 15 106 >120 
GroupNo. of ratsNo. of tumor cellsTumor occurrence %Death rate % (in 30 days)Death rate % (in 40 days)Survival (days)
Young control ratsa 20 105 100 20 100 32.7± 3.5 
Age-matched control ratsb 18 106 100 83 100 28.2± 2.8 
Cured rats (1st challenge)c 15 106 >120 
Cured rats (2nd challenge)d 15 106 >120 
Cured rats (3rd challenge)e 15 106 >120 
a

Female Wistar Furth rats with tumor inoculation at the age of 8 weeks, with the normal tumor dose of 105 viable tumor cells.

b

Untreated rats of the same age as the cured rats at the time of inoculation, without previous exposure to tumor.

c

These tumor-bearing rats cured by laser-ICG-GC treatment were challenged with 106 viable tumor cells 120 days after the initial inoculation.

d

Successfully treated rats from different experimental groups were challenged the second time after the first challenge.

e

Successfully treated rats from different experimental groups were challenged the third time after the second challenge.

We thank Scottye Davis for animal preparation.

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